Inhibition by ethylenediamine tetraacetate and restoration by Mn2+ and Ca2+ of oxygen-evolving activity in Photosystem II preparation from the thermophilic cyanobacterium,Synechococcus sp

The Photosystem II particles capable of evolving oxygen with ferricyanide as an electron acceptor were isolated from β-octylglucoside-solubilized thylakoid membranes of the thermophilic cyanobacterium Synechococcus sp. High and steady rates of oxygen evolution were observed only in the presence of 0.5% digitonin and 1 M sucrose. The activity was totally lost when the particles had been treated with 1 mM ethylenediamine tetraacetate for 1 min in a hypotonic medium. The treatment removed two out of four Mn atoms present per the Photosystem II reaction center in the particles. A significant rate of oxygen evolution was restored almost immediately after the addition of 5 mM MnCl₂ to the treated particles. The activity was also slowly and partially restored when the treated particles had been incubated with 5 mM CaCl₂. MgCl₂ was much less effective irrespective of the incubation time. The addition of MnCl₂ to the CaCl₂-restored particles increased the activity to a level which is significantly larger than the sum of the MnCl₂- and the CaCl₂-induced increases in the activity. The Mn- and Ca-effects were largely suppressed by the simultaneous addition of CaCl₂ and MnCl₂, respectively. It is concluded that both Mn²⁺ and Ca²⁺ are essential components of the water oxidation of photosynthesis, but the two cations support oxygen evolution through different mechanisms.     

Effects of calcium on the activities of cytosolic antioxidative enzymes and IAA oxidase during in vitro adventitious rooting of mung bean seedlings

The effects of Ca²⁺ on antioxidative enzymes and indole-3-acetic acid (IAA) oxidase during adventitious rooting were investigated in mung bean (Vigna radiata). CaCl₂ significantly promoted the formation and growth of adventitious roots. EGTA (a Ca²⁺ chelator) or ruthenium red (a Ca²⁺-channel blocker) significantly inhibited root formation and growth, but these inhibitory effects could be partially reversed by CaCl₂. Furthermore, inclusion of 5 mM CaCl₂ significantly increased superoxide dismutase (SOD) activity by 10% at 3 h and catalase (CAT) activity by an average of 29.6% at each time point. CaCl₂ decreased peroxidase (POD) activity by 9.4% and 21% at 12 and 24 h, respectively, and ascorbate peroxidase (APX) activity by an average of 13.9% at each time point. These CaCl₂-induced changes in enzymatic activities were similar to changes caused by indole-3-butyric acid (IBA). Treatment with EGTA or ruthenium red decreased SOD activity by an average of 18.4% and 15.2%, respectively; POD activity by 27.4% and 57.6%, respectively; APX activity by 10.3% and 15.6%, respectively; and CAT activity by 19.3% and 5.2%, respectively, when compared with CaCl₂. In addition, CaCl₂ increased IAA oxidase activity by an average of 5.5% beginning at 6 h, whereas EGTA significantly decreased IAA oxidase activity by 29.2%, 22.9%, and 13.5% at 6, 9, and 12 h, respectively. The inhibitory effects of EGTA could be partially suppressed by addition of CaCl₂. These results imply that the stimulative effect of Ca²⁺ on adventitious rooting is partially related to Ca²⁺-induced changes in the activities of antioxidative enzymes and IAA oxidase.     

Increase effected by calcium ion in the rate of oxygen evolution from preparations of Phormidium luridum

The presence of Ca²⁺ causes a twentyfold or greater increase in the rate of oxygen evolution by cell-free preparations of Phormidium luridum. The requirement for Ca²⁺ is specific; other divalent cations are much less effective or are inhibitory. The rate of the Hill reaction is maximal at 30 mM CaCl₂ in both detergent-free and Brij 35 preparations. The 3-(3,4-dichlorophenyl)-1,1-dimethylurea-insensitive component of oxygen-evolving activity in each preparation also shows the requirement for added Ca²⁺. This indicates that Ca²⁺ is acting close to the oxygen-evolving reaction center of Photosystem II. Defatted bovine serum albumin increases the rate of oxygen evolution in the detergent-free preparation, but does not compete with Ca²⁺, discounting fatty acid mediation of the effects of Ca²⁺. Neither excess Hill acceptor nor uncouplers of photophosphorylation diminish the stimulatory effects of Ca²⁺.     

Stabilization of calcium uptake in rat rod outer segments by taurine and ATP

Summary. Calcium ion (Ca²⁺) uptake was measured in rod outer segments (ROS) isolated from rat retina in the presence of varying concentrations of CaCl₂ in the incubation buffer (1.0–2.5 mM). It is known that taurine increases Ca²⁺ uptake in rat ROS in the presence of ATP and at low concentrations of CaCl₂ (Lombardini, 1985a); taurine produces no significant effects when CaCl₂ concentrations are increased to 1.0 and 2.5 mM. With the removal of both taurine and ATP, Ca²⁺ uptake in rat ROS increased significantly in the presence of 2.5 mM CaCl₂. Taurine treatment in the absence of ATP was effective in decreasing Ca²⁺ uptake at the higher levels of CaCl₂ (2.0 and 2.5 mM). Similar effects were observed with ATP treatment. The data suggest that taurine and ATP, alone or in combination, limit the capacity of the rat ROS to take up Ca²⁺ to the extent that a stable uptake level is achieved under conditions of increasing extracellular Ca²⁺, indicating a protective role for both agents against calcium toxicity. Received January 25, 2000/Accepted January 31, 2000     

Ca<Superscript>2+</Superscript> and CaM are Involved in NO- and H<Subscript>2</Subscript>O<Subscript>2</Subscript>-Induced Adventitious Root Development in Marigold

Our previous results have demonstrated that both nitric oxide (NO) and hydrogen peroxide (H₂O₂) are involved in the promotion of adventitious root development in marigold (Tagetes erecta L.). However, not much is known about the intricate molecular network of adventitious root development triggered by NO and H₂O₂. In this study, the involvement of calcium (Ca²⁺) and calmodulin (CaM) in NO- and H₂O₂-induced adventitious rooting in marigold was investigated. Exogenous Ca²⁺ was capable of promoting adventitious rooting, with a maximal biological response at 50 μM CaCl₂. Ca²⁺ chelators and CaM antagonists prevented NO- and H₂O₂-induced adventitious rooting, indicating that both endogenous Ca²⁺ and CaM may play crucial roles in the adventitious rooting induced by NO and H₂O₂. NO and H₂O₂ treatments increased the endogenous content of Ca²⁺ and CaM, suggesting that NO and H₂O₂ enhanced adventitious rooting by stimulating the endogenous Ca²⁺ and CaM levels. Moreover, treatment with Ca²⁺ enhanced the endogenous levels of NO and H₂O₂. Additionally, Ca²⁺ might be involved as an upstream signaling molecule for CaM during NO- and H₂O₂-induced rooting. Altogether, the results suggest that both Ca²⁺ and CaM are two downstream signaling molecules in adventitious rooting induced by NO and H₂O₂.     

Ca2+ and Mg2+-binding and a putative calmodulin type Ca2+-binding site in Synechococcus Photosystem II

Thylakoids and Photosystem II particles prepared from the cyanobacterium Synechococcus PCC 7942 washed with a HEPES/glycerol buffer exhibited low rates of light-induced oxygen evolution. Addition of either Ca²⁺ or Mg²⁺ to both thylakoids and Photosystem II particles increased oxygen evolution independently, maximal rates being obtained by addition of both ions. If either preparation was washed with NaCl, light induced O₂ evolution was completely inhibited, but re-activated in the same manner by Ca²⁺ and Mg²⁺ but to a lower level. In the presence of Mg²⁺, the reactivation of O₂ evolution by Ca²⁺ allowed sigmoid kinetics, implying co-operative binding. The results are interpreted as indicating that not only Ca²⁺, but also Mg²⁺, is essential for light-induced oxygen evolution in thylakoids and Photosystem II particles from Synechococcus PC 7942. The significance of the reactivation kinetics is discussed. Reactivation by Ca²⁺ was inhibited by antibodies to mammalian calmodulin, indicating that the binding site in Photosystem II may be analogous to that of this protein.Abbreviation HEPES n-2-Hydroxyethylpiperazine--2-ethane sulphonic acid     

Mn2+ reduces Yz + in manganese-depleted Photosystem II preparations

Manganese in the oxygen-evolving complex is a physiological electron donor to Photosystem II. PS II depleted of manganese may oxidize exogenous reductants including benzidine and Mn²⁺. Using flash photolysis with electron spin resonance detection, we examined the room-temperature reaction kinetics of these reductants with Y_z ⁺, the tyrosine radical formed in PS II membranes under illumination. Kinetics were measured with membranes that did or did not contain the 33 kDa extrinsic polypeptide of PS II, whose presence had no effect on the reaction kinetics with either reductant. The rate of Y_z ⁺ reduction by benzidine was a linear function of benzidine concentration. The rate of Y_z ⁺ reduction by Mn²⁺ at pH 6 increased linearly at low Mn²⁺ concentrations and reached a maximum at the Mn²⁺ concentrations equal to several times the reaction center concentration. The rate was inhibited by K⁺, Ca²⁺ and Mg²⁺. These data are described by a model in which negative charge on the membrane causes a local increase in the cation concentration. The rate of Y_z ⁺ reduction at pH 7.5 was biphasic with a fast 400 s phase that suggests binding of Mn²⁺ near Y_z ⁺ at a site that may be one of the native manganese binding sites.Abbreviations PS II Photosystem II - Y_D tyrosine residue in Photosystem II that gives rise to the stable Signal II EPR spectrum - Y_z tyrosine residue in Photosystem II that mediates electron transfer between the reaction center chlorophyll and the site of water oxidation - ESR electron spin resonance - DPC diphenylcarbazide - DCIP dichlorophenolindophenol     

A sustained increase in the intracellular Ca2+ concentration induces proteolytic cleavage of EAG2 channel

Voltage-gated EAG2 channel is abundant in the brain and enhances cancer cell growth by controlling cell volume. The channel contains a cyclic nucleotide-binding homology (CNBH) domain and multiple calmodulin-binding motifs. Here we show that a raised intracellular Ca²⁺ concentration causes proteolytic digestion of heterologously expressed and native EAG2 channels. A treatment of EAG2-expressing cells with the Ca²⁺ ionophore A23187 for 1 h reduces the full-length protein by ∼80% with a concomitant appearance of 30–35-kDa peptides. Similarly, a treatment with the Ca²⁺-ATPase inhibitor thapsigargin for 3 h removes 30–35-kDa peptides from ∼1/3 of the channel protein. Moreover, an incubation of the isolated rat brain membrane with CaCl₂ leads to the generation of fragments with similar sizes. This Ca²⁺-induced digestion is not seen with EAG1. Mutations in a C-terminal calmodulin-binding motif alter the degrees and positions of the cleavage. Truncated channels that mimic the digested proteins exhibit a reduced current density and altered channel gating. In particular, these shorter channels lack a rapid activation typical in EAG channels with more than 20-mV positive shifts in voltage dependence of activation. The truncation also eliminates the ability of EAG2 channel to reduce cell volume. These results suggest that a sustained increase in the intracellular Ca²⁺ concentration leads to proteolytic cleavage at the C-terminal cytosolic region following the CNBH domain by altering its interaction with calmodulin. The observed Ca²⁺-induced proteolytic cleavage of EAG2 channel may act as an adaptive response under physiological and/or pathological conditions.     

Inactivation of Ca2+-induced ciliary reversal by high-salt extraction in the cilia of Paramecium

Intracellular Ca²⁺ induces ciliary reversal and backward swimming in Paramecium. However, it is not known how the Ca²⁺ signal controls the motor machinery to induce ciliary reversal. We found that demembranated cilia on the ciliated cortical sheets from Paramecium caudatum lost the ability to undergo ciliary reversal after brief extraction with a solution containing 0.5 M KCl. KNO₃, which is similar to KCl with respect to chaotropic effect; it had the same effect as that of KCl on ciliary response. Cyclic AMP antagonizes Ca²⁺-induced ciliary reversal. Limited trypsin digestion prevents endogenous A-kinase and cAMP-dependent phosphorylation of an outer arm dynein light chain and induces ciliary reversal. However, the trypsin digestion prior to the high-salt extraction did not affect the inhibition of Ca²⁺-induced ciliary reversal caused by the high-salt extraction. Furthermore, during the course of the high-salt extraction, some axonemal proteins were extracted from ciliary axonemes, suggesting that they may be responsible for Ca²⁺-induced ciliary reversal.     

Ca2+-induced lateral phase separations in phosphatidic acid-phosphatidylcholine membranes

The effects of Ca²⁺ on phosphatidic acid-phosphatidylcholine membranes have been studied using phospholipid spin labels. ESR spectra of spin-labeled phosphatidic acid-phosphatidylcholine membranes and phosphatidic acid-spin-labeled phosphatidylcholine membranes are exchange-broadened immediately upon addition of CaCl₂. These changes directly and conclusively indicate Ca²⁺-induced clustering of spin-labeled phosphatidylcholine and aggregation of spin-labeled phosphatidic acid bridged by Ca²⁺-chelation in the binary phopholipid membranes. In the Ca²⁺-chelated aggregates, the motions of the alkyl chains of phosphatidic acid are greatly reduced and the lipid molecules are more closely packed. The clusters and aggregates are formed in patches and the sizes are dependent on the fractions. Ba²⁺ and Sr²⁺ induce the lateral phase separations to the same extent as Ca²⁺. Mg²⁺ is also effective but to a lesser extent. In acid solutions (pH 5.5), the Ca²⁺-induced lateral phase separations are of slightly lesser extent than in alkaline solution (pH 7.9). These results are compared with those for phosphatidylserine-phosphatidylcholine membranes reported previously and necessary conditions for the lateral phase separations are discussed.     

The melatonin receptor antagonist luzindole induces Ca2+ mobilization,reactive oxygen species generation and impairs trypsin secretion in mouse pancreatic acinar cells

BackgroundIn this work we studied the effects of the melatonin receptor-antagonist luzindole (1 μM–50 μM) on isolated mouse pancreatic acinar cells.MethodsChanges in intracellular free-Ca²⁺ concentration, reactive oxygen species production and trypsin secretion were analyzed.ResultsLuzindole induced increases in [Ca²⁺]_i that diminished CCK-8 induced Ca²⁺ mobilization, compared with that observed when CCK-8 was applied alone. Treatment of cells with thapsigargin (1 μM), in the absence of Ca²⁺ in the extracellular medium, evoked a transient increase in [Ca²⁺]_i. The additional incubation of cells with luzindole (10 μM) failed to induce further mobilization of Ca²⁺. In the presence of luzindole a concentration-dependent increase in ROS generation was observed that decreased in the absence of Ca²⁺ or by pretreatment of cells with melatonin (100 μM). Incubation of pancreatic acinar cells with luzindole (10 μM) impaired CCK-8-induced trypsin secretion. Melatonin was unable to revert the effect of luzindole on CCK-8-induced trypsin secretion.ConclusionThe melatonin receptor-inhibitor luzindole induces Ca²⁺-mediated pro-oxidative conditions and impairment of enzyme secretion, which creates a situation in pancreatic acinar cells that might compromise their function.General significanceThe effects of luzindole that we have observed, might be unspecific and could mislead the observations when it is used to study the actions of melatonin on the gland. Another possibility is that melatonin receptors exhibit a basal or agonist-independent activity in pancreatic acinar cells, which might be modulated by melatonin or luzindole.     

Control of Ca2+ efflux and cyclic AMP by 5-hydroxytryptamine and dopamine in abalone gill.

5-Hydroxytrptamine increased the rate of Ca²⁺ efflux and the concentration of endogenous cyclic AMP in abalone gill in both 10 mM and 50 mM CaCl₂ concentrations externally. Dopamine decreased the rate of Ca²⁺ efflux in 50 mM CaCl₂ but slightly increased the efflux rate in 10 mM CaCl₂. At both external Ca²⁺ concentrations, dopamine increased the endogenous cyclic AMP concentration in the gill. 5-Hydroxytryptamine but not dopamine was found to activate adenylate cyclase in broken cell preparations of abalone gill. Cyclic AMP-dependent protein kinase activity was also demonstrated in homogenate fractions of abalone gill. It is suggested that both Ca²⁺ and cyclic AMP act as second messengers in the response of abalone gill to 5-hydroxytryptamine and dopamine.     

Relation between microsecond reduction kinetics of photooxidized chlorophyll aII (P-680) and photosynthetic water oxidation

The reduction phases of chlorophyll a⁺_II (P-680⁺) in the microsecond range have been studied in O₂-evolving Photosystem II particles from Synechococcus sp. and in spinach subchloroplasts. (1) In selected Photosystem II preparations only approx. 15% of chlorophyll a⁺_II is reduced under repetitive excitation in the microsecond time-range (approx. 85% are reduced in the nanosecond time-range). (2) The size of the microsecond fraction varies as a function of the flash number given to dark-adapted samples, suggesting a correlation to the oxidation states of the O₂-evolving complex (S-states). The oscillatory pattern closely follows the concentration of S₂ + S₃. (3) The microsecond decay can be deconvoluted into three exponential phases with half-life times of approx. 5, 35 and 200 μs. It is the amplitude of the 35 μs phase which depends on S₂ + S₃. Therefore, the 35 μs phase (approx. 10% under repetitive excitation) is connected with water oxidation. (4) Considerably higher values of the μs fraction (up to 50%) reported in former publications were probably due to Photosystem II centers which were inactive in O₂ evolution.     

Polar lobe formation and cytokinesis in fertilized eggs of Ilyanassa obsoleta. II. Large bleb formation caused by high concentrations of exogenous calcium ions

Fertilized eggs of the mollusk Ilyanassa obsoleta (Nassarius obsoletus) form large blebs resembling polar lobes within 12 min of exposure to solutions of isotonic CaCl₂, whereas control eggs in sea water remain spherical. Under identical conditions, fertilized eggs of the sea urchin, Strongylocentrotus purpuratus, an organism which normally does not form polar lobes, do not form blebs upon exposure to solutions of isotonic CaCl₂. The calcium-induced blebbing in Ilyanassa still occurs if other cations such as Na⁺, Mg²⁺, or Mn²⁺ are present in addition to Ca²⁺, but not if comparable concentrations of K⁺ are present. Cytochalasin B prevents the calcium-induced blebbing, whereas colchicine does not. Cytokinesis in both Ilyanassa and Strongylocentrotus and normal polar lobe formation in Ilyanassa appear to require exogenous K⁺ but not exogenous Ca²⁺. Preliminary electron microscopy of Ilyanassa eggs exposed to isotonic solutions of CaCl₂ has shown microfilaments in the cortical cytoplasm in the region of the bleb constriction but no microfilaments in spherical control eggs in sea water. These data suggest that high concentrations of exogenous Ca²⁺ trigger the polymerization and contraction of a ring of microfilaments in the cortical cytoplasm of the Ilyanassa egg which results in the formation of a lobelike bleb of cytoplasm. The observation that K⁺ antagonizes this Ca²⁺-induced blebbing has led to the formulation of a theory which postulates that the ratio of intracellular Ca²⁺ to intracellular K⁺ is critical in the control of polar lobe formation and cytokinesis.     

Extracellular calcium is involved in stomatal movement through the regulation of water channels in broad bean

Involvement of extracellular Ca²⁺ in stomatal movement through the regulation of water channels was investigated in broad bean (Vicia faba L.). Leaf peels were first incubated to open stomata, and then transferred to buffers in the presence of different CaCl₂ concentrations. Stomatal status was observed under magnification and stomatal aperture (pore width/length) was measured. Stomatal closure was significantly induced and aperture oscillation occurred at lower extracellular concentrations of calcium ([Ca²⁺]_ext), while at higher concentrations, no significant change in stomatal aperture was observed, which was similar to the response recorded with HgCl₂. Lower [Ca²⁺]_ext-induced stomatal closure could be reversed using depolarizing buffer. It is suggested that lower [Ca²⁺]_ext regulates water channels through an indirect way and at higher concentrations, extracellular Ca²⁺ is involved in regulating stomatal aperture by directly influencing water channels to retard aperture change.     

Potentiation of bleomycin cytotoxicity by membrane-interacting drugs and increased calcium ions

The cytotoxic effect of the antitumor antibiotic peplomycin (PEP), a new member of bleomycin group antibiotics, toward HeLa cells and mouse FM3A cells is enhanced by some membrane-interacting drugs such as verapamil, persantin, prenylamine, chlorpromazine and anafranil. The enhancing action of verapamil is selective to this group antibiotics, since it does not potentiate the cytotoxic effects of vincristine, adriamycin, mitomycin C, cis-diamminedichloroplatinum(II) and macromomycin. An enhanced PEP cytotoxicity has been also demonstrated by the treatment of cells in the presence of increased CaCl₂. This enhancing effect of increased CaCl₂ is prevented by the Ca²⁺ transport inhibitor ruthenium red. Since these membrane-directed drugs have been shown to affect Ca²⁺ metabolism, we conclude that potentiation of PEP cytotoxicity by these drugs is mediated by an increase in intracellular Ca²⁺.     

Effects of calcium on growth and leaf ion concentrations of Gossypium hirsutum grown in saline hydroponic culture

The optimum Ca²⁺ concentration for growth of cotton (Gossypium hirsutum cv. Acala SJ-2) was in the range 1 to 15 mol m^–3 for plants growing in hydroponic culture with 100–150 mol m^–3 NaCl. Most saline (but not sodic) soils contain higher Ca²⁺ concentrations. CaCl₂ was inhibitory to the growth of cotton above 20–50 mol m^–3. Increasing concentrations of Ca²⁺ in the range 0–2 mol m^–2 drastically reduced Na⁺ accumulation in the leaves. As CaCl₂ concentrations were increased above the optimum for growth there was a further reduction in leaf Na⁺ accumulation, but this was more than offset by increased leaf Ca²⁺ and Cl^– concentrations. Leaf K⁺ concentrations were not much affected by changes in external CaCl₂ concentrations. The response of Mg²⁺ varied from an increase to a decrease with increasing external CaCl₂ and was influenced by nutritional status. There was no evidence that high Ca²⁺ caused a deficiency of Mg²⁺ in cotton. Except for Cl^–, whose concentrations tended to decrease initially and then increase as the CaCl₂ concentration increased, the anions were largely unaffected by changes in external CaCl₂.     

Interactive effects of Ca2+ and NaCl salinity on the growth of two tomato genotypes differing in Ca2+ use efficiency

Two tomato (Lycopersicon esculentum Mill.) lines differing in Ca²⁺ use efficiency (Ca²⁺ use efficient line 113 and Ca²⁺ use inefficient line 67) were subjected to salinity treatments in two separate experiments to determine whether they differed in salt tolerance. In experiment I, three NaCl and two CaCl₂ treatments were imposed. The Na⁺ concentrations were 1.1, 100 and 150 mM and the Ca²⁺ concentrations were either 1.51 or 10 mM. In experiment II, one NaCl and three Ca²⁺ treatments (as CaCl₂ or CaSO₄) were imposed. The treatments consisted of 150 mM NaCl at either 1.51 mM CaCl₂, 10 mM CaCl₂, or 10 mM CaSO₄. Response to treatments was determined by analysis of growth parameters (shoot and root dry weights, plant height, and root length). Shoot and root dry weight, and root length were depressed as salinity increased in plants lacking additional Ca²⁺. No significant differences in salt tolerance were detected between the two tomato lines after 24 d of salinity treatment. An important finding of this study was that root growth and length appeared to be more sensitive to the effect of CaCI₂ treatment alone and to the effects of CaCl₂ × NaCl treatments. This suggests that over the long term, both root growth and root length may be more sensitive indicators of salinity effects than shoots. Supplemental CaCl₂ had no ameliorative effect on NaCl stress in shoot growth. The inability of Ca²⁺ to counter Cl⁻ entry or toxicity may account for the lack of amelioration. Additional Ca²⁺ as CaSO₄ improved shoot growth of plants exposed to 150 mM NaCl. In contrast, root growth and length were improved by 10 mM Ca²⁺ as either CaCl₂ or CaSO₄.     

Effects of Calcium and Calmodulin Antagonists on Chilling Stress-Induced Proline Accumulation in <Emphasis Type="Italic">Jatropha curcas</Emphasis> L.

Regulation of proline accumulation in plants under chilling stress remains unclear. In this paper, we treated Jatropha curcas seedlings under chilling stress with exogenous calcium chloride (CaCl₂), the plasma membrane Ca²⁺-channel blocker lanthanum chloride (LaCl₃), calmodulin antagonists, chlorpromazine (CPZ), and trifluoperazine (TFP) and investigated the effects of calcium and calmodulin (CaM) on proline accumulation and chilling tolerance. The results showed that CaCl₂ treatment significantly enhanced chilling stress-induced proline accumulation. CaCl₂ also induced an almost immediate and rapid increase of Δ¹-pyrroline-5-carboxylate synthetase (P5CS) and glutamate dehydrogenase activities, the key enzymes in the glutamate pathway of proline biosynthesis, and up-regulated P5CS expression, but it decreased the activity of proline dehydrogenase (ProDH), a key enzyme of proline degradation, and inhibited ProDH expression. Treatment with LaCl₃, CPZ, and TFP exhibited the opposite effects to those by CaCl₂ treatment. Moreover, CaCl₂, LaCl₃, CPZ, and TFP had little effect on the activities of ornithine aminotransferase and arginase, the key enzymes in the ornithine pathway of proline biosynthesis. These results indicated that Ca²⁺-CaM might be involved in signal transduction events, leading to proline accumulation in J. curcas seedlings under chilling stress, and that Ca²⁺-induced proline accumulation is a combined result of the activation of the glutamate pathways of proline biosynthesis and the simultaneous inhibition of the proline degradation pathway. In addition, CaCl₂ treatment increased tissue vitality, decreased the content of the lipid peroxidation product malondialdehyde (MDA), and alleviated electrolyte leakage in J. curcas seedlings under chilling stress, indicating that exogenous Ca²⁺ can enhance chilling tolerance, and proline might be a key factor in this increased chilling tolerance.