PPF1 inhibits programmed cell death in apical meristems of both G2 pea and transgenic Arabidopsis plants possibly by delaying cytosolic Ca2+ elevation

PPF1 encodes a putative calcium ion carrier that affects the flowering time of transgenic Arabidopsis by modulating Ca(2+) storage capacities in chloroplasts of a plant cell. In the current work, we found that differential expression of PPF1 might affect processes of programmed cell death (PCD) since DNA fragmentation was detected in senescencing apical buds of long day-grown G2 pea (Pisum sativum L.) plants, but was not in non-senescencing short day-grown counterparts at all growth stages. An animal inhibitor of caspase-activated DNase (ICAD) homologue was detected in short day-grown plant continuously throughout the whole experiment and only in early stages of long day-grown pre-floral G2 pea apical buds. DNA fragmentation was significantly inhibited in apical meristems of transgenic Arabidopsis that over-expressed the PPF1 gene when compared to that of either wild-type control or to PPF1 (-) plants. The expression of ICAD-like protein decreased to undetectable level at 45 dpg in apical tissues of PPF1 (-) Arabidopsis, which was much earlier than that found in PPF1 (+) or wild-type controls. In epidermal cells of PPF1 (-) plants, we recorded significantly earlier calcium transient prior to PCD. We suggest that the expression of PPF1, a chloroplast localized Ca(2+) ion channel may inhibit programmed cell death in apical meristems of flowering plants by keeping a low cytoplasmic calcium content that might inhibit DNA fragmentation in plant cells.     

定位于类囊体膜的PPF1在转基因拟南芥中的表达影响叶绿体的发育

PPF1是一个与植物营养生长相关的基因。它编码的产物可能是一个膜蛋白并与拟南芥叶绿体中的类囊体蛋白ALB3有很高的同源性。免疫电镜分析表明PPF1蛋白同样主要定位于类囊体膜 ,而且在短日照G2豌豆开花两周后仍发育良好的叶绿体中有很高的表达 ,在长日照豌豆同时期非正常叶绿体中丰度非常低。对转基因拟南芥和野生型植株的叶片衰老进程比较发现 ,PPF1在拟南芥中的过量表达可以延缓叶片的衰老 ,而用PPF1反义mRNA抑制拟南芥中的同源基因ALB3则明显加快叶片衰老速度。对转基因拟南芥的超微结构分析显示 ,PPF1在拟南芥中过量表达时 ,转基因植株的叶绿体比野生型植株的叶绿体大并含有更多的基粒和基质类囊体膜 ;相反 ,反义PPF1表达抑制其在拟南芥中的同源物时 ,转基因植株的叶绿体比野生型植株的叶绿体小并含有较少的基粒和发育较差的类囊体膜系统。这些数据表明叶绿体的发育状况与PPF1或拟南芥同源物ALB3的表达水平呈正相关。我们的结果提示PPF1基因可能通过控制叶绿体的发育状况来调节植物的发育。     

PPF1 may suppress plant senescence via activating TFL1 in transgenic Arabidopsis plants

Senescence, a sequence of biochemical and physiological events, constitutes the final stage of development In higher plants and is modulated by a variety of environmental factors and intemal factors. PPF1 possesses an important biological function in plant development by controlling the Ca2 storage capacity within chloroplasts. Here we show that the expression of PPF1 might play a pivotal role in negatively regulating plant senescence as revealed by the regulation of overexpression and suppression of PPF1 on plant development. Moreover, TFL1, a key regulator in the floral repression pathway, was screened out as one of the downstream targets for PPF1 in the senescence-signaling pathway. Investigation of the senescence-related phenotypes in PPF1(-) tfl1-1 and PPF1( ) tfl1-1 double mutants confirmed and further highlighted the relation of PPF1 with TFL1 in tranegenic plants. The activation of TFL1 expression by PPF1 defines an important pathway possibly essential for the negative regulation of plant senescence in transgenic Arabidopsis.     

Comparative analysis of plant and animal calcium signal transduction element using plant full-length cDNA data

We obtained 32K full-length cDNA sequence data from the rice full-length cDNA project and performed a homology search against NCBI GenBank data. We have also searched homologs of Arabidopsis and other plants' genes with the databases. Comparative analysis of calcium ion transport proteins revealed that the genes specific for muscle and nerve calcium signal transduction systems (VDCC, IP3 receptor, ryanodine receptor) are very different in animals and plants. In contrast, Ca elements with basic functions in cell responses (CNGC, iGlu receptor, Ca(2+)ATPase, Ca2+/Na(+)-K+ ion exchanger) are basically conserved between plants and animals. We also performed comparative analyses of calcium ion binding and/or controlling signal transduction proteins. Many genes specific for muscle and nerve tissue do not exist in plants. However, calcium ion signal transduction genes of basic functions of cell homeostasis and responses were well conserved; plants have developed a calcium ion interacting system that is more direct than in animals. Many species of plants have specifically modified calcium ion binding proteins (CPK, CRK), Ca2+/phospholipid-binding domains, and calcium storage proteins.     

TPK1 is a vacuolar ion channel different from the slow-vacuolar cation channel

TPK1 (formerly KCO1) is the founding member of the family of two-pore domain K(+) channels in Arabidopsis (Arabidopsis thaliana), which originally was described following expression in Sf9 insect cells as a Ca(2+)- and voltage-dependent outwardly rectifying plasma membrane K(+) channel. In plants, this channel has been shown by green fluorescent protein fusion to localize to the vacuolar membrane, which led to speculations that the TPK1 gene product would be a component of the nonselective, Ca(2+) and voltage-dependent slow-vacuolar (SV) cation channel found in many plants species. Using yeast (Saccharomyces cerevisiae) as an expression system for TPK1, we show functional expression of the channel in the vacuolar membrane. In isolated vacuoles of yeast yvc1 disruption mutants, the TPK1 gene product shows ion channel activity with some characteristics very similar to the SV-type channel. The open channel conductance of TPK1 in symmetrically 100 mM KCl is slightly asymmetric with roughly 40 pS at positive membrane voltages and 75 pS at negative voltages. Similar to the SV-type channel, TPK1 is activated by cytosolic Ca(2+), requiring micromolar concentration for activation. However, in contrast to the SV-type channel, TPK1 exhibits strong selectivity for K(+) over Na(+), and its activity turned out to be independent of the membrane voltage over the range of +/-80 mV. Our data clearly demonstrate that TPK1 is a voltage-independent, Ca(2+)-activated, K(+)-selective ion channel in the vacuolar membrane that does not mediate SV-type ionic currents.     

Calcium binding to the epidermal growth factor homology region of bovine protein C

A high affinity calcium binding site that is independent of the gamma-carboxyglutamic acid-rich amino-terminal region, has been demonstrated in bovine protein C, as well as in the other vitamin K-dependent proteins (except prothrombin) involved in blood coagulation. gamma-Carboxyglutamic acid-independent calcium binding in protein C is required for its rapid activation by the thrombin-thrombomodulin complex. We have now isolated a Ca2+-binding fragment from a tryptic digest of bovine protein C. The isolated fragment contains the two domains that are homologous to the epidermal growth factor precursor from the light chain of protein C, and a small disulfide bound peptide derived from the heavy chain. The isolated fragment bound 1 mol of Ca2+/mol of protein with a dissociation constant (Kd) of approximately 1 x 10(-4) M. This is similar to the Kd previously determined for binding of a single Ca2+ ion to protein C lacking the gamma-carboxyglutamic acid region. Immunochemical evidence indicated that Ca2+ binding induced a conformational change both in protein C lacking the gamma-carboxyglutamic acid region and in the isolated fragment.     

Calcium specificity of the antigen-induced channels in rat basophilic leukemia cells

Ion channels, activated upon IgE-Fc epsilon receptor aggregation by specific antigen, were studied in micropipet-supported lipid bilayers. These bilayers were reconstituted with purified IgE-Fc epsilon receptor complex and the intact 110-kDa channel-forming protein, both isolated from plasma membranes of rat basophilic leukemia cells (line RBL-2H3). In order to identify the current carrier through these ion channels and to determine their ion selectivity, we investigated the currents flowing through the IgE-Fc epsilon receptor gated channels in the presence of a gradient of Ca2+ ions. Thus, the solution in which the micropipet-supported bilayer was immersed contained 1.8 mM CaCl2, while the interior of the micropipet contained 0.1 microM Ca2+ (buffered with EGTA). Both solutions also contained 150 mM of a monovalent cation chloride salt (either K+ or Na+). The currents induced upon specific aggregation of the IgE (by either antigen or anti-IgE antibodies) were examined over a range of potentials imposed on the bilayer. The type of conductance event most frequently observed under the employed experimental conditions was a channel that has a slope conductance of 3 pS and a reversal potential practically identical with the calculated value for the reversal potential of calcium (134 +/- 11 mV in the presence of sodium, 125 +/- 13 mV in the presence of potassium). These results indicate that this channel is highly selective for calcium against the monovalent cations sodium and potassium. This same channel has a conductance of 4-5 pS in the presence of symmetrical solutions containing only 100 mM CaCl2 and 8 pS in the presence of 0.5 M NaCl with no calcium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on the stereoselective effects of a novel 5-HT2 receptor antagonist on contractile responses of rat aorta

The effect of the enantiomers of a novel 5-HT2 receptor antagonist, (+/-)-(1R,3S)-1-[2-[4-[3-(p-fluorophenyl)-1-indanyl]-piperazinyl] ethyl]-2-imidazolidinone, was studied on serotonin (5-HT), noradrenaline (NA), potassium (K+), and calcium (Ca2+)-induced contractions in isolated rat thoracic aorta. The enantiomers shifted the 5-HT, NA, K+, and Ca2+ concentration-response curves to the right in a concentration-dependent manner and depressed the maximal contractile responses. The (+)-enantiomer was a far more potent inhibitor of 5-HT-induced contractions than the (-)-enantiomer. The (+)-enantiomer and phentolamine, both at 10(-6) M, had equal inhibitory effects on NA-evoked contractions. The (+)-enantiomer was again more potent inhibiting NA-induced contractions than the (-)-enantiomer. Both enantiomers had an equieffective inhibitory effect on K+ and Ca2(+)-induced contractions. The results show that the 5-HT and alpha-adrenoceptor antagonism of the two enantiomers is stereoselective, the (+)-enantiomer being more potent than the (-)-enantiomer. In contrast the enantiomers had equal, nonstereoselective inhibitory effects on K+ and Ca2(+)-evoked contractions.     

Inositol 1,4,5-trisphosphate-triggered Ca2+ release from bovine adrenal medullary secretory vesicles

The effect of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and calcium ionophore A23187 on Ca2+ release from bovine adrenal medullary secretory vesicles and microsomes was examined. Ins(1,4,5)P3 released 3.5 nmol of Ca2+/mg protein from secretory vesicles and 1.5 nmol of Ca2+/mg protein from microsomes as measured by a Ca2(+)-selective electrode. However, A23187 promoted Ca2+ uptake into vesicles while releasing Ca2+ from microsomes. Ins(1,4,5)P3-induced Ca2+ release from secretory vesicles was rapid, but the released Ca2+ was absorbed within 3 min during which the Ins(1,4,5)P3-releasable pools were refilled. The in situ calcium content of secretory vesicle measured by atomic absorption spectrometry was 112 +/- 6.3 nmol/mg protein indicating the potential importance of secretory vesicles as an intracellular Ca2+ store. The high Ca2(+)-buffering capacity of secretory vesicles is presumed to be due to the high Ca2(+)-binding capacity of chromogranin A, the major intravesicular protein, which has calsequestrin-like properties.     

Multiple calcium channels in synaptosomes: voltage dependence of 1,4-dihydropyridine binding and effects on function

The voltage dependence of binding of the calcium channel antagonist, (+)-[3H]PN200-110, to rat brain synaptosomes and the effects of dihydropyridines on 45Ca2+ uptake have been investigated. Under nondepolarizing conditions (+)-[3H]PN200-110 binds to a single class of sites with a Kd of 0.07 nM and a binding capacity of 182 fmol/mg of protein. When the synaptosomal membrane potential was dissipated either by osmotic lysis of the synaptosomes or by depolarization induced by raising the external K+ concentration, there was a decrease in affinity (approximately 7-fold) with no change in the number of sites. The effects of calcium channel ligands on 45Ca2+ uptake by synaptosomes have been measured as a function of external potassium concentration, i.e., membrane potential. Depolarization led to a rapid influx of 45Ca2+ whose magnitude was voltage-dependent. Verapamil (100 microM) almost completely inhibited calcium uptake at all potassium concentrations studied. In contrast, the effects of dihydropyridines (2 microM) appear to be voltage-sensitive. At relatively low levels of depolarization (10-25 mM K+) nitrendipine and PN200-110 completely inhibited 45Ca2+ influx, whereas the agonist Bay K8644 slightly potentiated the response. At higher K+ concentrations an additional dihydropyridine-insensitive component of calcium uptake was observed. These results provide evidence for the presence of dihydropyridine-sensitive calcium channels in synaptosomes which may be activated under conditions of partial depolarization.     

Optical measurements of Na-Ca-K exchange currents in intact outer segments isolated from bovine retinal rods

The properties of Na-Ca-K exchange current through the plasma membrane of intact rod outer segments (ROS) isolated from bovine retinas were studied with the optical probe neutral red. Small cellular organelles such as bovine ROS do not offer an adequate collecting area to measure Na-Ca-K exchange currents with electrophysiological techniques. This study demonstrates that Na-Ca-K exchange current in bovine ROS can be measured with the dye neutral red and dual-wavelength spectrophotometry. The binding of neutral red is sensitive to transport of cations across the plasma membrane of ROS by the effect of the translocated cations on the surface potential of the intracellular disk membranes (1985. J. Membr. Biol. 88: 249-262). Electrogenic Na+ fluxes through the ROS plasma membrane were measured with a resolution of 10(5) Na+ ions/ROS per s, equivalent to a current of approximately 0.01 pA; maximal electrogenic Na-Ca-K exchange flux in bovine ROS was equivalent to a maximal exchange current of 1-2 pA. Electrogenic Na+ fluxes were identified as Na-Ca-K exchange current based on a comparison between electrogenic Na+ flux and Na(+)-stimulated Ca2+ release with respect to flux rate, Na+ dependence, and ion selectivity. Neutral red monitored the net entry of a single positive charge carried by Na+ for each Ca2+ ion released (i.e., monitored the Na-Ca-K exchange current). Na-Ca-K exchange in the plasma membrane of bovine ROS had the following properties: (a) Inward Na-Ca-K exchange current required internal Ca2+ (half-maximal stimulation at a free Ca2+ concentration of 0.9 microM), whereas outward Na-Ca-K exchange current required both external Ca2+ (half-maximal stimulation at a free Ca2+ concentration of 1.1 microM) and external K+. (b) Inward Na-Ca-K exchange current depended in a sigmoidal manner on the external Na+ concentration, identical to Na(+)-stimulated Ca2+ release measured with Ca(2+)-indicating dyes. (c) The neutral red method was modified to measure Ca(2+)-activated K+ fluxes (half-maximal stimulation at 2.7 microM free Ca2+) via the Na-Ca-K exchanger in support of the notion that the rod Na-Ca exchanger is in effect a Na-Ca-K exchanger. (d) Competitive interactions between Ca2+ and Na+ ions on the exchanger protein are described.     

Extracellular calcium participates in responses to acetylcholine in Xenopus oocytes

We tested the contribution of extracellular calcium (Ca2+) to membrane electrical responses to acetylcholine (ACh) in native Xenopus oocytes. Removal of Cao caused a decrease in both the rapid (D1) and the slow (D2) chloride currents that comprise the common depolarizing response to ACh in native oocyte. The effect of Ca2+o removal on the muscarinic response was mimicked by the addition of 1 mM Mn2+, an effective antagonist of calcium influx, though not by antagonists of voltage-sensitive calcium channels. When oocytes were challenged with ACh in Ca2(+)-free medium, subsequent addition of 1.8 mM CaCl2 resulted in a rapid, often transient, depolarizing current. Similarly to the Ca2+o-dependent component of membrane electrical responses, the Ca2(+)-evoked current was reversibly abolished by Mn2+, though not by antigonists of voltage-sensitive calcium channels. Depletion of cellular calcium potentiated the Ca2(+)-evoked current, implying negative feedback of calcium channels by calcium. Injection of 10-100 fmol of inositol 1,4,5-trisphosphate (IP3) resulted in a two-component depolarizing current. IP3 injection promoted the appearance of Ca2+o-evoked current that was significantly potentiated by previous calcium depletion. We suggest that activation of cell-membrane muscarinic receptors causes opening of apparently voltage-insensitive and verapamil or diltiazem-resistant calcium channels. These channels may be activated by IP3 or its metabolites, which increase following the activation of cell membrane receptors coupled to a phospholipase C. The channels may be identical to receptor-operated channels described in other model systems.     

The evaluation of the role of endogenous Ca-dependent regulators and protein kinases in activating and inhibiting ion-transport ATPases]

The data on hormonal regulation of ATP-driving ion pumps are contradictory depending on the object used: whether native cells or isolated membranes. To eliminate this contrariety, we studied the ion transporting ATPases in saponin-permeabilized cells in the presence of all endogenous regulators. In permeabilized erythrocytes we obtained the presence of Ca(2+)-dependent activation of Ca(2+)-ATPase by factor(s) not affected by calmodulin antagonist R24571. We obtained also Ca(2+)-dependent activation and inhibition of Na+,K(+)-ATPase. At a concentration of Mg(2+)-ions corresponding to the intracellular level (370 microM), the 0.5-0.7 microM Ca(2+)-activated Na+,K(+)-ATPase (up to 3-fold), whereas the 1-5 microM Ca2+ inhibited it. The cyclic AMP (10(-5) M) inhibited or eliminated Ca(2+)-dependent activation. The decrease in Mg(2+)-ion concentration to 50 microM eliminated the activation and strengthened the inhibition, which reached 100% at the 1-2 microM Ca2+ concentration. The washing of membranes with EGTA eliminated Ca2+ effects on Na+,K(+)-ATPase. These data suggest that the ion-transporting ATPases are activated or inhibited by Ca(2+)-dependent regulators whose activities may be changed by protein kinase catalysed phosphorylation.     

Growth retardation of plants transformed by overexpression of NtFtsZ1-2 in Tobacco

We transformed tobacco plants (Mcotiana tabacum L, Xanthi) by introducing a sense construct ofNtFtsZ1-2. This tobacco nuclear gene encodes a chloroplast-localized homologue of FtsZ, the bacterial cell-division protein. The overexpressing plants contained enlarged chloroplasts in their leaf mesophyll cells. In the T₁ progeny, we observed three different phenotypes: 1 ) plants with cells containing many small chloroplasts, which was the same as for wild-type plants; 2) plants in which the celts contained one to three enlarged chloroplasts (severe type); and 3) plants whose cells contained a combination of many small chloroplasts and one to three enlarged chloroplasts (intermediate type). The outward appearance of the severe and intermediate types of transgenic plants did not differ noticeably from the wild-types. However, the severe-type plants were most retarded in their growth under both high- and low-light conditions, followed by the intermediate-types. Under medium levels of light, the two types of transgenic plants exhibited growth rates comparable to that of the wild types. Based on the overall results, we suggest that many small chloroplasts, rather than a few large chloroplasts, are required for efficient use of light energy in the mesophyll cells.     

In planta regulation of the Arabidopsis Ca(2+)/H(+) antiporter CAX1

Vacuolar localized Ca(2+)/H(+) exchangers such as Arabidopsis thaliana cation exchanger 1 (CAX1) play important roles in Ca(2+) homeostasis. When expressed in yeast, CAX1 is regulated via an N-terminal autoinhibitory domain. In yeast expression assays, a 36 amino acid N-terminal truncation of CAX1, termed sCAX1, and variants with specific mutations in this N-terminus, show CAX1-mediated Ca(2+)/H(+) antiport activity. Furthermore, transgenic plants expressing sCAX1 display increased Ca(2+) accumulation and heightened activity of vacuolar Ca(2+)/H(+) antiport. Here the properties of N-terminal CAX1 variants in plants and yeast expression systems are compared and contrasted to determine if autoinhibition of CAX1 is occurring in planta. Initially, using ionome analysis, it has been demonstrated that only yeast cells expressing activated CAX1 transporters have altered total calcium content and fluctuations in zinc and nickel. Tobacco plants expressing activated CAX1 variants displayed hypersensitivity to ion imbalances, increased calcium accumulation, heightened concentrations of other mineral nutrients such as potassium, magnesium and manganese, and increased activity of tonoplast-enriched Ca(2+)/H(+) transport. Despite high in planta gene expression, CAX1 and N-terminal variants of CAX1 which were not active in yeast, displayed none of the aforementioned phenotypes. Although several plant transporters appear to contain N-terminal autoinhibitory domains, this work is the first to document clearly N-terminal-dependent regulation of a Ca(2+) transporter in transgenic plants. Engineering the autoinhibitory domain thus provides a strategy to enhance transport function to affect agronomic traits.     

Cd2+ transport and storage in the chloroplast of Euglena gracilis

Euglena gracilis lacks a plant-like vacuole and, when grown in Cd2+-containing medium, 60% of the accumulated Cd2+ is located inside the chloroplast. Hence, the biochemical mechanisms involved in Cd2+ accumulation in chloroplast were examined. Percoll-purified chloroplasts showed a temperature-sensitive uptake of the free 109Cd2+ ion. Kinetics of the uptake initial rate was resolved in two components, one hyperbolic and saturable (Vmax 11 nmol 109Cd2+ min(-1) mg protein (-1), Km 13 microM) and the other, linear and non-saturable. 109Cd2+ uptake was not affected by metabolic inhibitors or illumination. Zn2+ competitively inhibited 109Cd2+ uptake (Ki 8.2 microM); internal Cd2+ slightly inhibited 109Cd2+ uptake. Cadmium was partially and rapidly released from chloroplasts. These data suggested the involvement of a cation diffusion facilitator-like protein. Chloroplasts isolated from cells grown with 50 microM CdCl2 (ZCd50 chloroplasts) showed a 1.6 times increase in the uptake Vmax, whereas the Km and the non-saturable component did not change. In addition, Cd2+ retention in chloroplasts correlated with the amount of internal sulfur compounds. ZCd50 chloroplasts, which contained 4.4 times more thiol-compounds and sulfide than control chloroplasts, retained six times more Cd2+. The Cd2+ storage-inactivation mechanism was specific for Cd2+, since Zn2+ and Fe3+ were not preferentially accumulated into chloroplasts.     

Purification and characterization of a novel 12,000-Da calcium binding protein from smooth muscle.

A new low molecular weight calcium binding protein, designated 12-kDa CaBP, has been isolated from chicken gizzard using a phenyl-Sepharose affinity column followed by ion-exchange and gel filtration chromatographies. The isolated protein was homogeneous and has a molecular weight of 12,000 based on sodium dodecyl sulfate-gel electrophoresis. The amino acid composition of this protein is similar to but distinct from other known low molecular weight Ca2+ binding proteins. Ca2+ binding assays using Arsenazo III (Sigma) indicated that the protein binds 1 mol of Ca2+/mol of protein. The 12-kDa CaBP underwent a conformational change upon binding Ca2+, as revealed by uv difference spectroscopy and circular dichroism studies in the aromatic and far-ultraviolet range. Addition of Ca2+ to the 12-kDa CaBP labeled with 2-p-toluidinylnaphthalene-6-sulfonate (TNS) resulted in a sevenfold increase in fluorescence intensity, accompanied by a blue shift of the emission maximum from 463 to 445 nm. Hence, the probe in the presence of Ca2+ moves to a more nonpolar microenvironment. Like calmodulin and other related Ca2+ binding proteins, this protein also exposes a hydrophobic site upon binding calcium. Fluorescence titration with Ca2+ using TNS-labeled protein revealed the presence of a single high affinity calcium binding site (kd approximately 1 x 10(-6) M).     

Changes of Ca2+ -ATPase Activities in Cell of Rice Seed lings During the Enhancement of Chilling Resistance Induced by Cold and Salt Pretreatment

The changes of Ca2+ -ATPase activities of plasmolemma, and tonoplast membrane in roots and leaf chloroplasts in rice ( Oryza sativa L. ) seedlings were investigated for exploring the mechanism of cross adaptation to different stresses in the plants during the enhancement of chilling resistance induced by cold and salt pretreatment. The results indicated that the chilling resistance of rice seedlings was enhanced markedly by cold and salt pretreatment, but this enhancement was inhibited by Ca2+-chelate ethyleneglycol-bis-(β-aminoethyl ether) N, N-tetraacetic acid (EGTA) and the calmodulin inhibitor chlorpromazine (CPZ), it showed the calcium messenger system was involved in the course of chilling resistance formation. The Ca2+ -ATPase activity of root plasmolemma and tonoplast membrane as well as the Fe(CN)63- reduction in root plasmolemma in nonpretreated seedlings were declined markedly during the chilling stress. The Ca2+ -ATPase activities of plasmolemma, tonoplast membrane and chloroplasts as well as the Fe(CN)63- reduction of plasmolemma were enhanced by cold pretreatment. The activities of Ca2+ -ATPase and Fe(CN)63- reduction of plasmolemma, as compared with nonpretreated seedlings has increased by 86.80% and 93.93% respectively. The effect of salt pretreatmerit on the Ca2+ -ATPase activities of plasmolemma and chloroplast as well as Fe(CN)63- reduction of plasmolemma were similar to the effect of cold pretreatment. Although the Ca2+ -ATPase activity of tonoplast membrane was declined by salt pretreatment, the activity was none the less markedly higher than that of the nonpretreated seedlings. It showed that there was stronger ability of maintaining calcium homeostasis in the seedlings following two pretreatment. The results displayed that the enhancement of chilling resistance in rice seedlings with cold and salt pretreatment might be related to the effective activation of Ca2+ -ATPase in two pretreatment seedlings, because the activated Ca2+ -ATPase could bring back rapidly the raised cytoplasmic Ca2+ concentration from chilling stress to the state of calcium homeostasis, leading to the maintenance of normal functioning of the calcium messenger system and physiological metabolism. It seems that the adapated mechanism to chilling stress in two seedlings with cold and salt pretreatment was similar.     

SCD-1基因过表达对鸭子宫上皮细胞钙离子和脂质含量的效应

硬脂酰辅酶A去饱和酶-1 (Stearoyl-CoA desaturase-1,SCD-1)是催化单不饱和脂肪酸合成的关键性蛋白酶,Ca²⁺是生物体内重要阳离子,在生物体内发挥着重要作用。为探讨SCD-1基因与脂质指标和钙离子含量之间的关联性,通过构建pcDNA3.1(+)+SCD-1+Flag真核过表达载体和培养鸭子宫上皮细胞并共转染,通过载体上Flag标签检测SCD-1基因的过表达量,用Fluo-3/AM钙离子荧光标记法检测Ca²⁺浓度,用脂质指标试剂盒检测细胞内的脂质含量。结果表明,鸭SCD-1基因过表达量与甘油三酯(TG)和高密度脂蛋白胆固醇(HDL-C)含量呈负相关,与Ca²⁺浓度、总胆固醇(TC)含量、极低密度脂蛋白胆固醇(VLDL-C)含量和低密度脂蛋白胆固醇(LDL-C)含量呈正相关;Ca²⁺与TG、LDL-C和HDL-C的含量呈正相关,与TC和VLDL-C含量呈负相关,研究结果揭示了SCD-1基因过表达能够调控鸭子宫上皮细胞中Ca²⁺浓度和脂质合成与转运。