Detoxification of dissolved SO2 (bisulfite) by terricolous mosses

BACKGROUND AND AIMS: The widespread calcifuge moss Pleurozium schreberi is moderately tolerant of SO2, whereas Rhytidiadelphus triquetrus is limited to calcareous soils in regions of the UK that were strongly affected by SO2 pollution in the 20th century. The proposition that tolerance of SO2 by these terricolous mosses depends on metabolic detoxification of dissolved bisulfite was investigated. METHODS: The capacities of the two mosses to accelerate loss of bisulfite from aqueous solutions of NaHSO3 were studied using DTNB [5, 5-dithio-(2-nitrobenzoic acid)] to assay bisulfite, and HPLC to assay sulfate in the incubation solutions. Incubations were performed for different durations, in the presence and absence of light, at a range of solution pH values, in the presence of metabolic inhibitors and with altered moss apoplastic Ca2+ and Fe3+ levels. KEY RESULTS: Bisulfite disappearance was markedly stimulated in the light and twice as great for R. triquetrus as for P. schreberi. DCMU, an inhibitor of photosynthetic electron chain transport, significantly reduced bisulfite loss. CONCLUSIONS: Bisulfite (SO2) tolerance in these terricolous mosses involves extracellular oxidation using metabolic (photo-oxidative) energy, passive oxidation by adsorbed Fe3+ (only available to the calcifuge) and probably also internal metabolic detoxification.     

Regional Calcicoly in the Moss Rhytidiadelphus triquetrus: Survival and Chemistry of Transplant at a Formerly SO2-polluted Site with Acid Soil

The moss Rhytidiadelphus triquetrus has become increasinglyrestricted to calcareous soils in the countryside around Londonsince the middle of the present century. It has been suggestedthat this is due either to high SO₂ pollution in earlier decadesor continuing acidification of precipitation and soils. Intactsamples of Rhytidiadelphus carpet (25 x 25 cm) were transplantedfrom a chalk rendzina at Watlington Hill to clearings in Pleuroziumschreberi swards on acid clay soil at Windsor Forest to determinewhether conditions are currently unfavourable for Rhytidiadelphus. Some transplants included calcareous soil introduced alongwith the mosses. Tissue concentration of exchangeable and intracellularCa, Mg, K and Al were measured in intervals over 381 d. TheRhytidiadelphus transplant remained healthy after 33 monthson acid soil. Shoot exchangeable Mg and K rose rapidly on transplantationto Windsor Forest while exchangeable Ca fell significantly onlyafter 1 year. Intracellular K was unaffected by transplantation.Tissue cation levels were little affected by the nature of thesoil beneath the transplant but instead reflected the grosschemistry of the receiver site. This indicates that bryophytenutrient content is mainly influenced by precipitation and throughfallminerals. Soil chemistry may influence feather mosses via indirectpathway such as leaf leachates or dust. Aluminum levels werenear the detection limits and did not change significantly inthe transplants. The absence of Rhytidiadelphus from acid soilsin Windsor Forest is Probably due to historical factors suchas previous episode of high SO₂ concentrations.Copyright 1993,1999 Academic Press Rhytidiadelphus triquetrus, Pleuroziums schreberi, SO₂, acid rain, aluminum, calcium, magnesium, potassium     

The development of potential screens based on shoot calcium and iron concentrations for the evaluation of tolerance in Egyptian genotypes of white lupin (Lupinus albus L.) to limed soils

European cultivars of white lupin (Lupinus albus L.) grow poorly in limed or calcareous soils. However, Egyptian genotypes are grown successfully in highly calcareous soil and show no stress symptoms. To examine their physiological responses to alkaline soil and develop potential screens for tolerance, three experiments were conducted in limed and non-limed (neutral pH) soil. Measurements included net CO2 uptake, and the partitioning of Fe2+ and Fe3+ and soluble and insoluble Ca in stem and leaf tissue. Intolerant plants showed clear symptoms of stress, whereas stress in the Egyptian genotypes and in L pilosus Murr. (a tolerant species) was less marked. Only the intolerant plants became chlorotic and this contributed to their reduced net CO2 uptake in the limed soil. In contrast, Egyptian genotypes and L pilosus showed no change in net CO2 uptake between the soils. The partitioning of Ca and Fe either resulted from the stress responses, or was itself a stress response. L pilosus and some Egyptian genotypes differed in soluble Ca concentrations compared with the intolerant cultivars, although no significant difference was apparent in the Ca partitioning of the Egyptian genotype Giza 1. In a limed soil, Giza 1 maintained its stem Fe3+ concentration at a level comparable with that of plants grown in non-limed soil, whereas stem [Fe3+] of an intolerant genotype increased. Gizal increased the percentage of plant Fe that was Fe2+ in its leaf tissue under these conditions; that of the intolerant genotype was reduced. The potential tolerance of the Egyptian genotypes through these mechanisms and the possibility of nutritional-based screens are discussed.     

The effect of nitrogen nutrition on cluster root formation and proton extrusion by Lupinus albus

Nitrogen nutrition can influence cluster root formation in many wild species, but the effect of N form on cluster root formation and root exudation by white lupin is not known. In a solution culture study, we examined the effect of N nutrition (ammonium, nitrate, both or N2 fixation) on cluster root formation and H+ extrusion by white lupin plants under deficient and adequate P supply. The number of cluster roots increased greatly when plants were supplied with I microM P compared with 50 microM P, the increase being 7.8-fold for plants treated with (NH4)2SO4, 3-fold for plants treated with KNO3 and NH4NO3, and 2-4-fold for N2-fixing plants. Under P deficiency. NH4+-N supply resulted in production of a greater number and biomass of cluster roots than other N sources. Dry weight of cluster roots was 30 % higher than that of non-cluster roots in P-deficient plants treated with (NH4)2SO4 and NH4NO3. In plants treated with sufficient P (50 microM), the weight of non-cluster roots was approx. 90 % greater than that of cluster roots. Both total (micromol per plant h(-1)) and specific (micromol g(-1) root d. wt h(-1)) H+ extrusions were greatest from roots of plants supplied with (NH4)2SO4, followed by those supplied with NH4NO3 and N2 fixation, whereas plants receiving KNO3 had negative net H+ extrusion between the third and fifth week of growth (indicating uptake of protons or release of OH- ions). The rate of proton extrusion by NH4+-N-fed plants was similar under P-deficient and P-sufficient conditions. In contrast, proton exudation by N2-fixing plants and KNO3-treated plants was ten-fold greater under P deficiency than under P sufficiency. In comparison with P deficiency, plants treated with 50 microM P had a significantly higher concentration of P in roots, shoots and youngest expanded leaves (YEL). Compared with the N2 fixation and KNO3 treatments, total N concentration was highest in roots, shoots and YEL of plants supplied with (NH4)2SO4 and NH4NO3, regardless of P supply. Under P deficiency, K concentrations in roots decreased at all N supplies, especially in plants treated with (NH4)2SO4 and NH4NO3, which coincided with the greatest H+ extrusion at these P and N supplies. In conclusion, NH4-N nutrition stimulated cluster root formation and H+ extrusion by roots of P-deficient white lupin.     

Calcium in plants

Calcium is an essential plant nutrient. It is required for various structural roles in the cell wall and membranes, it is a counter-cation for inorganic and organic anions in the vacuole, and the cytosolic Ca2+ concentration ([Ca2+]cyt) is an obligate intracellular messenger coordinating responses to numerous developmental cues and environmental challenges. This article provides an overview of the nutritional requirements of different plants for Ca, and how this impacts on natural flora and the Ca content of crops. It also reviews recent work on (a) the mechanisms of Ca2+ transport across cellular membranes, (b) understanding the origins and specificity of [Ca2+]cyt signals and (c) characterizing the cellular [Ca2+]cyt-sensors (such as calmodulin, calcineurin B-like proteins and calcium-dependent protein kinases) that allow plant cells to respond appropriately to [Ca2+]cyt signals.     

Encoding specificity in plant calcium signalling: hot-spotting the ups and downs and waves

Calcium ions function as intracellular second messengers in regulating a plethora of cellular processes from acclimative stress responses to survival and programmed cell death. The generation of specificity in Ca2+ signals is dependent on influx and efflux from the extracellular milieu, cytosol and intracellular organelles. One aspect of plant Ca2+ signalling that is currently attracting a great deal of interest is how 'Ca2+-signatures', specific spatio-temporal changes in cytosolic-free Ca2+, encode the necessary information to bring about this range of physiological responses. Here, current information is reviewed on how Ca2+-signatures are generated in plant cells and how stimulus-specific information can be encoded in the form of Ca2+-signatures.     

Fe2+-Induced Lysis and Lipid Peroxidation of Chromaffin Granules

Chromaffin granules, the catecholaminergic storage granules from adrenal chromaffin cells, lysed in 10(-9)-10(-7) M Fe2+. Lysis was accompanied by the production of malondialdehyde which results from lipid peroxidation. Both chromaffin granule lysis and malondialdehyde production were inhibited by the free radical trapping agent butylated hydroxytoluene but not by catalase and/or superoxide dismutase. The results suggest that lysis resulted from a direct transfer of electrons from Fe2+ to a component of the chromaffin granule membrane without the participation of either superoxide or hydrogen peroxide and may have resulted from lipid peroxidation. In some experiments, ascorbate alone induced chromaffin granule lysis which was inhibited by EDTA, EGTA, or deferoxamine. The lysis was probably caused by trace amounts of reducible polyvalent cation. Lysis sometimes occurred when Ca2+ was added with EGTA (10 microM free Ca2+ concentration) and was consistently observed together with malondialdehyde production in the presence of Ca2+, EGTA, and 10 microM Fe2+ (total concentration). The apparent Ca2+ dependency for chromaffin granule lysis and malondialdehyde production was probably caused by a trace reducible polyvalent ion displaced by Ca2+ from EGTA and not by a Ca2+-dependent reaction involving the chromaffin granule.     

Aluminium toxicity in rye (Secale cereale): root growth and dynamics of cytoplasmic Ca2+ in intact root tips

Aluminium (Al) toxicity in rye (Secale cereale L.), an Al-resistant crop, was examined by measuring root elongation and cytoplasmic free activity of calcium ([Ca2+]cyt) in intact root apical cells. Measurement of [Ca2+]cyt, was achieved by loading a Ca2+-sensitive fluorescent probe. Fluo-3/AM ester, into root apical cells followed by detection of intracellular fluorescence using a confocal laser scanning microscope. After 20 min of exposure to 50 microM Al (pH 4-2) a slight increase in [Ca2+]cyt of root apical cells was observed, while the response of [Ca2+]cyt to 100 microM Al (pH 4.2) was faster and larger ([Ca2+]cyt increased by 46% in 10 min). Increases in [Ca2+]cyt were correlated with inhibition of root growth, generally measurable after 2 h. Addition of 400 microM malic acid (pH 4.2) largely ameliorated the effect of 100 microM Al on [Ca2+]cyt in root apical cells and protected root growth from Al toxicity. These results suggest that an increase in [Ca2+]cyt in root apical cells in rye is an early effect of Al toxicity and is followed by the secondary effect on root elongation.     

Nitric oxide signalling in plants: interplays with Ca2+ and protein kinases

Much attention has been paid to nitric oxide (NO) research since its discovery as a physiological mediator of plant defence responses. In recent years, newer roles have been attributed to NO, ranging from root development to stomatal closure. The molecular mechanisms underlying NO action in plants are just begun to emerge. The currently available data illustrate that NO can directly influence the activity of target proteins through nitrosylation and has the capacity to act as a Ca2+-mobilizing intracellular messenger. The interplay between NO and Ca2+ has important functional implications, expanding and enriching the possibilities for modulating transduction processes. Furthermore, protein kinases regulated through NO-dependent mechanisms are being discovered, offering fresh perspective on processes such as stress tolerance.     

Do we know the absolute values of intracellular free calcium concentration?

More than 20 years ago, it was shown that the addition of EGTA increases the affinity of the plasma membrane Ca2+ pump for Ca2+ by an order of magnitude. The left-hand shift of Ca2+-dependencies in the presence of EGTA has been also documented in studies of the sarcoplasmic reticulum Ca2+ pump, mitochondrial Ca2+-transporter as well as Ca2+-binding by calmodulin and troponin C. These data allow us to hypothesise that this effect is caused by an admixture of di- and trivalent cations possessing high affinity for EGTA and interacting with Ca2+-transporting and binding proteins. Here, we propose that polyvalent cations affect the estimation of absolute values of free intracellular Ca2+ concentration. Indeed, EGTA sharply increases the apparent affinity of the fluorescent Ca2+ indicators quin-2 and fluo-3 for Ca2+. The impact of polyvalent cations on Ca2+ measurement was further confirmed by the study showing the high sensitivity of Ca2+-induced fluo-3 fluorescence to Mn2+, Fe2+, Cu2+, and Co2+ seen in the absence of EGTA.     

Growth,net photosynthesis and leaf nutrient status of <Emphasis Type="Italic">Fagus crenata</Emphasis> seedlings grown in brown forest soil acidified with H<Subscript>2</Subscript>SO<Subscript>4</Subscript> or HNO<Subscript>3</Subscript> solution

To obtain basic information for evaluating critical loads of acid deposition for protecting Japanese beech forests, growth, net photosynthesis and leaf nutrient status of Fagus crenata seedlings grown for two growing seasons in brown forest soil acidified with H₂SO₄ or HNO₃ solution were investigated. The whole-plant dry mass of the seedlings grown in the soil acidified by the addition of H₂SO₄ or HNO₃ solution was significantly less than that of the seedlings grown in the control soil not supplemented with H⁺ as H₂SO₄ or HNO₃ solution. However, the degrees of reduction in the whole-plant dry mass and net photosynthetic rate of the seedlings grown in the soil acidified by the addition of H⁺ as H₂SO₄ solution at 100 mg l^–1 on the basis of air-dried soil volume (S-100 treatment) were greater than those of the seedlings grown in the soil acidified by the addition of H⁺ as HNO₃ solution at 100 mg l^–1 (N-100 treatment). The concentrations of Al and Mn in the leaves of the seedlings grown in the S-100 treatment were significantly higher than those in the N-100 treatment. A positive correlation was obtained between the molar ratio of (Ca+Mg+K)/(Al+Mn) in the soil solution and the relative whole-plant dry mass of the seedlings grown in the acidified soils to that of the seedlings grown in the control soil. Based on the results, we concluded that the negative effects of soil acidification due to sulfate deposition are greater than those of soil acidification due to nitrate deposition on growth, net photosynthesis and leaf nutrient status of F. crenata, and that the molar ratio of (Ca+Mg+K)/(Al+Mn) in soil solution is a suitable soil parameter for evaluating critical loads of acid deposition in efforts to protect F. crenata forests in Japan.     

Functional Analysis of the Genotypic Differences in Response of Pea (<i>Pisum sativum</i> L.) to Calcareous-Induced Iron Deficiency

Lime-induced iron chlorosis is a major nutritional disorder causing severe plant growth and yield reduction in the calcareous soils of Tunisia. The understanding the behavior of key metabolic functions of peas on calcareous soils, the identification of useful traits of tolerance, and the exploration of the genotypic differences in response to this constraint remain the most efficient approaches due to their coast, environmental benefits, and sustainability. For this purpose, a greenhouse experiment was conducted on three pea genotypes (Alexandra: Alex, Douce de provence: DP, and Merveille de Kelvedon: MK) cultivated on calcareous soil (Fe-deficient) and fertile soil (control). Plant growth, SPAD index, iron nutrition and distribution, photosynthesis, and antioxidant enzymes were deeply analyzed to discriminate genotypic differences. Calcareous-induced iron deficiency reduced SPAD index, plant growth, net photosynthesis, and tissue Fe content against a significant stimulation of the oxidative stress indicators, H₂O₂ and Malondialdehyde (MDA). Moreover, we reported a significant induction of SOD and CAT activity in shoots and roots of the Alexandra genotype. Fe use efficiency increased on calcareous soil and clearly discriminated the studied genotypes. Alexandra genotype was found to be the most tolerant to lime-induced iron chlorosis. This genotype protects its tissues against oxidative stress by stimulating enzyme activities (SOD and CAT) and develops significant efficiency of Fe uptake, translocation to shoots and use when cultivated on calcareous soil.     

Spatial Variability of Soil Chemical Properties of a Prairie–Forest Transition in Louisiana

Woody plant expansion, particularly eastern red cedar (Juniperus virginiana L.), has been a major threat to Louisiana calcareous prairies. Previous studies have shown that woody plant expansion into grasslands is associated with an increase in soil heterogeneity. We studied the within site spatial variability and among site differences of surface (0–15 cm depth) soil pH, electrical conductivity (EC), and Mehlich III extractable Ca, Mg, K, Fe and Mn from three remnant prairie-forest associations in Winn Parish, Louisiana. The prairie soil was consistently basic (pH > 7.0) and the forest soil was acidic (pH < 7.0) while the transition soil was neutral (pH = 7.0). A nonparametric statistical test for the equality of medians among sites showed the median values of the soil attributes differed (α = 0.05) except for soil Ca and Fe. The similarity in Ca concentration among sites was attributed to the calcareous parent material common to the three sites. Geostatistical analysis showed that spatial dependence was expressed over a range of 20–30 m for most of the soil attributes considered. Semivariogram shapes were similar among sites, suggesting the greater control of soil parent material on the observed spatial soil pattern. Shorter range of variation emerged only for soil pH when soil data from the forest and transition were deleted, indicating the scaling characteristics of soil pH and its susceptibility to plant induced changes. It is concluded that soil pH can be used as an index to determine prairie-forest boundary, and to access the impact of eastern red cedar on these and similar sites derived from calcareous parent material. Further, results from this study can be used for designing future ecological studies within the prairie by taking the soil spatial variability into account.     

Inward currents and increases in cytosolic Ca2+ concentration induced by cyclic ADP-ribose in turtle olfactory receptor cells

In olfactory receptor cells, it is well established that cyclic AMP (cAMP) and inositol-1,4,5-trisphosphate (IP(3)) act as second messengers during odor responses. In previous studies, we have shown that cAMP-increasing odorants induce odor responses even after complete desensitization of the cAMP-mediated pathway. These results suggest that at least one cAMP-independent pathway contributes to the generation of odor responses. In an attempt to identify a novel second messenger, we investigated the possible role of cyclic ADP-ribose (cADPR) in olfactory transduction. Turtle olfactory receptor cells were isolated using an enzyme-free procedure and loaded with fura-2/AM. The cells responded to dialysis with cADPR with an inward current and an increase of the intracellular Ca(2+) concentration, [Ca(2+)](i). Flooding of cells with 100 microM cADPR from the pipette also induced an inward current without changes in [Ca(2+)](i) in Na(+)-containing and Ca(2+)-free Ringer solution. In an Na(+)-free and Ca(2+)-containing Ringer solution, cADPR induced only a small inward current with a concomitant increase in [Ca(2+)](i). Inward currents and increases in [Ca(2+)](i) induced by cADPR were completely inhibited by removal of both Na(+) and Ca(2+) from the outer solution. The experiments suggest that cADPR activates a cation channel at the plasma membrane, allowing inflow of Na(+) and Ca(2+) ions. The magnitudes of the inward current responses to cAMP-increasing odorants were greatly reduced by prior dialyses of a high concentration of cADPR or 8-bromo-cyclic ADP-ribose (8-Br-cADPR), an antagonist. It is possible that the cADPR-dependent pathway contributes to the generation of olfactory responses.     

The Growth Response of Ecotypes of Holcus lanatus L. from Different Soil Types in Northwestern Europe to Phenolic Acids

Abstract: Phenolics are often discussed in relation to either allelopathy or to herbivory. This work, however, was undertaken to determine if phenolic acids benefit the growth of plants in very acid soils. We here report racial differences in the phenolic acid concentrations of the important plant species found in five sites within Central Scotland which covered a wide range in soil acidity from very acid (organic peats) to mildly acid (calcareous), and describe the racial differences in the growth response of Holcus lanatus L. to phenolic acids with increasing acidity. The total concentrations of phenolic acids in the ecotypes of important species were correlated with the total concentrations found in their respective soils. In general, the most phenolic-rich ecotypes of the five came from the organic acid soils (Flanders Moss [FM] and Sheriffmuir [SMB]). However, with the exception of ferulic acid which was a major component of both acid soils and their associated vegetation, individual simple phenolic acids extracted from either plants or soils were not consistently correlated. The addition of dry plant material collected from the five sites (0.5 g plant material 100 g^-1 soil) to the acid-organic Flanders Moss (FM) soil stimulated the growth of two ecotypes of Holcus (acid-mineral Sheriffmuir [SMM], calcareous Kinloch Rannoch [KR]) but the same litter addition in non-organic, less acidic or calcareous soil inhibited growth of these ecotypes. In hydroponic solutions, growth response of Flanders Moss (FM) and Kinloch Rannoch (KR) to pH and phenolic acid mixtures was interdependent: in acid solutions (pH 4.0) but not at pH 6.5 root elongation rates (RER) of both ecotypes, and shoot elongation rates (SER) of Flanders Moss (FM), increased after treatment with a mixture of seven commonly occurring phenolic acids.     

Influence of Norway spruce seedlings on the nutrient availability in mineral soil and forest floor material

A greenhouse experiment was performed to evaluate the effect of Norway spruce (Picea abies (L.) Karst.) seedlings on net nutrient availability in five different growing media containing F- or H-layer and mineral soil originating from a haplic podzol in northern Sweden. The initial total amounts of eight nutrient elements (N, K, P, Ca, Mg, Mn, Fe, Zn) and exchangeable amounts of the same elements were analyzed in pots with or without spruce seedlings. In the planted pots seedling nutrient uptake was also estimated. After 26 weeks, higher net nutrient availability with seedlings was found in 25 out of the 40 (62%) growing media and nutrient element combinations. A positive seedling effect on net nutrient availability might be explained by rhizodeposition stimulating the soil microorganism activity and accelerating the weathering of minerals or by seedling roots promoting the nutrient providing processes through changes in soil chemical and physical properties. Nitrogen availability was primarily affected by what part of the forest floor the growing medium contained although the positive response to seedling presence was apparent. The positive net availability response of P, Ca, Mg, Mn, Fe and Zn to seedling presence was on the other hand relatively strong. In the case of P, K, and Zn the growing medium composition (if the F- and H-layer was pure or mixed with mineral soil) was also an important factor for the estimated net availability. Pure F-and H-layer provided greater P- and K-availability while the availability of Zn increased when mineral soil was added. The influence of growing plants ought to be considered when soil samples are used for assessing the nutrient availability.     

Effects of [CO2] and nitrogen fertilization on soils planted with ponderosa pine

The effects of six years treatment with elevated [CO₂] (350, 525, and 700 μl l^-1) and nitrogen (N) (0, 10, and 20 g N m^-2 yr^-1) on soils, soil solution, and CO₂ efflux in an open-top chamber study with ponderosa pine (Pinus ponderosa Laws.) are described. The clearest [CO₂] effect was in year 6, when a pattern of lower soil N concentration and higher C/N ratio with elevated [CO₂] emerged. Statistically significant effects of elevated [CO₂] on soil total C, extractable P, exchangeable Mg²⁺, exchangeable Ca²⁺, base saturation, and soil solution HCO₃ ^- and NO₃ ^- were also found in various treatment combinations and at various times; however, these effects were inconsistent among treatments and years, and in many cases (P, Mg²⁺, Ca²⁺, base saturation) reflected pre-treatment differences. The use of homogenized buried soil bags did not improve the power to detect changes in soil C and N or help resolve the inconsistencies in soil C patterns. Nitrogen fertilization had the expected negative effects on exchangeable Ca²⁺, K⁺, and Mg²⁺ in year 6, presumably because of increased NO₃ ^- leaching, but had no consistent effect on soil C, N, or extractable P. This revised version was published online in June 2006 with corrections to the Cover Date.     

Type-3 ryanodine receptor involved in Ca2+-induced Ca2+ release and transmitter exocytosis at frog motor nerve terminals

Ca(2+)-induced Ca2+ release (CICR) occurs in frog motor nerve terminals after ryanodine receptors (RyRs) are primed for activation by conditioning large Ca2+ entry. We studied which type of RyR exists, whether CICR occurs without conditioning Ca2+ entry and how RyRs are primed. Immunohistochemistry revealed the existence of RyR3 in motor nerve terminals and axons and both RyR1 and RyR3 in muscle fibers. A blocker of RyR, 8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8) slightly decreased rises in intracellular Ca2+ ([Ca2+]i) induced by a short tetanus (50 Hz, 1-2s), but not after treatment with ryanodine. Repetitive tetani (50 Hz for 15s every 20s) produced repetitive rises in [Ca2+]i, whose amplitude overall waxed and waned. TMB-8 blocked the waxing and waning components. Ryanodine suppressed a slow increase in end-plate potentials (EPPs) induced by stimuli (33.3 Hz, 15s) in a low Ca2+, high Mg2+ solution. KN-62, a blocker of Ca(2+)/calmoduline-activated protein kinase II (CaMKII), slightly reduced short tetanus-induced rises in [Ca2+]i, but markedly the slow waxing and waning rises produced by repetitive tetani in both normal and low Ca2+, high Mg2+ solutions. Likewise, KN-62, but not KN-04, an inactive analog, suppressed slow increases in EPP amplitude and miniature EPP frequency during long tetanus. Thus, CICR normally occurs weakly via RyR3 activation by single impulse-induced Ca2+ entry in frog motor nerve terminals and greatly after the priming of RyR via CaMKII activation by conditioning Ca2+ entry, thus, facilitating transmitter exocytosis and its plasticity.