保卫细胞钙信号的研究进展

钙(Ca2+)是多种信号途径的第二信使。Ca2+成像技术的成熟和发展为显示保卫细胞胞 质Ca2+浓度（［Ca2+］cyt）的分布及外界刺激引起［Ca2+］cyt的变化模式提供了很好的研究工具,关于细胞内外Ca2+库释放Ca2+的机制也有了较清楚的认识。拟南芥突变体的研究使Ca2+ 信号上游分子及其排序更加明确,［Ca2+］cyt增加下游的磷酸化和去磷酸化 过程也是气孔关闭必需的生理过程。     

抗寒剂CR-4 对冬小麦幼苗质膜钙泵（Ca2 +-ATPase） 的稳定作用

通过磷酸铈沉淀的细胞化学观察揭示,常温下生长的冬小麦幼苗的Ca2+ -ATP酶活性主要定位在质膜上,同时,水浸种和抗寒剂浸种的小麦质膜Ca2+ -ATP酶活性没有差异。然而,小麦幼苗经-7℃冰冻处理12小时和24小时后,则表现明显的区别：水浸种的小麦幼苗质膜Ca2+ -ATP酶活性明显下降,直至完全失活,细胞的精细结构也同时被破坏;而经抗寒剂浸种的小麦幼苗质膜Ca2+ -ATP酶仍维持较高的活性,细胞结构也保持完整,显示抗寒剂对质膜Ca2+ -ATPase酶起着明显的稳定作用。     

槲皮素诱导Hep G2自噬与胞内游离Ca2+浓度的变化相关

槲皮素具有诱导细胞自噬、抑制肿瘤细胞增殖等抗癌功能,但其诱导细胞自噬的分子机制还不太清楚. 本文通过激光共聚焦显微镜观察槲皮素对Hep G2细胞自噬的影响; Fluo-3 AM和Cyto-IDTM Green Detection Reagent染色标记, 流式细胞术测定了槲皮素对Hep G2细胞内游离钙离子浓度［Ca2+］_i 及Ca2＋螯合剂BAPTA-AM对自噬水平的影响. 探讨了槲皮素诱导人肝癌细胞 Hep G2自噬过程中［Ca2+］i的变化. 结果表明, 在槲皮素较低浓度范围内(0 ～ 50 μg/mL), 可明显抑制Hep G2细胞增殖, 并以剂量依赖方式诱导细胞自噬. 同时发现,槲皮素刺激Hep G2细胞可使［Ca2+］i明显增加, 进而促进自噬. 而当胞内Ca2＋螯合剂 BAPTA-AM存在时, 细胞的自噬水平受到一定的抑制. 这些结果表明,细胞内［Ca2+］i的升高可促进自噬, ［Ca2+］_i 的降低可能会抑制自噬. Hep G2细胞自噬与细胞内游离钙离子浓度的变化有关系.     

Auxin induces an increase of Ca2+ concentration in the cytosol of wheat leaf protoplasts

Auxin addition to protoplasts isolated from leaves of 6-day-old wheat seedlings (Triticum aestivum L. cv. Kadett) induced a rapid increase in the cytosolic calcium concentration [Ca2+]cyt. The shifts in [Ca2+]cyt were detected by use of fluorescence microscopy in single protoplasts loaded with the calcium binding tetra[acetoxymethyl]ester of the fluorescent dye, Fura 2. Addition of the synthetic auxin naphthyl acetic acid, 1-NAA, induced an increase in [Ca2+]cyt within 5-10s, while the physiologically non-active analogue, 2-NAA, did not. The amplitude of calcium increase depended on the concentration of 1-NAA. Since the process was affected by different concentrations of Ca2+ in the external medium, and since the calcium channel blockers (nifedipine and verapamil) postponed and inhibited the reaction, it is suggested that auxin primarily activates Ca2+-permeable channels in the plasma membrane. In the presence of low external calcium concentration (0.1 mM), 5 mM LiCl almost totally blocked the increase in [Ca2+]cyt, indicating a possible involvement of tonoplast Ca2+-channels in the auxin-induced [Ca2+]cyt shift. Thus, calcium signalling induced by auxin involves both external and internal Ca2+ pools.     

正颤蚓的生长发育及繁殖生物学的研究

在实验室饲养的条件下,研究了正颤蚓在15℃、20℃、25℃水温的生长发育和繁殖过程。结果表明正颤蚓从其受精卵在蚓茧内发育,孵出生长到产卵,在15℃水温需100—123d,在20℃水温需70—90d,在25℃水温需48—64d。蚓自蚓茧内孵出后的165d生长期内,其15℃、20℃、25℃水温的体重生长回归方程分别是W=6.87/［1+136.48     

Ca(2)+-stimulated exocytosis in maize coleoptile cells

Changes in membrane capacitance (C(m)) after photolysis of the caged Ca(2)+ compound dimethoxynitrophenamine were studied in protoplasts from maize coleoptiles. Changes in C(m) values resulting from increased concentrations of free Ca(2)+ in the cytoplasm ([Ca(2)+](cyt)) were interpreted as representing changes in [Ca(2)+](cyt)-sensitive exocytosis and endocytosis. A continuous increase in [Ca(2)+](cyt) resulted in a sigmoidal increase in C(m) values with a half-maximal concentration at approximately 1 microM. The steep increase in C(m) values was followed by a variable slow phase in changing C(m) values. When [Ca(2)+](cyt) increased at a rate of 0.6 micromol L(-)(1) sec(-)(1), the initial steep increase in C(m) values lasted approximately 5 to 10 sec. During this time, protoplasts increased in surface area by approximately 2.5%. The biphasic dynamics of [Ca(2)+](cyt)-stimulated increases in C(m) values can be described by a kinetic model containing two pools of vesicles with two [Ca(2)+](cyt)-sensitive steps in the exocytotic pathway.     

Influence of Elevated CO2 Concentration and Nitrogen Source on Photosynthetic Traits in the Invasive Species Eupatorium adenophorum (Asteraceae)

Increases in the concentration of atmospheric CO2 and plant invasion are two important problems that face humans worldwide. In some plants, exposure to a short term elevated concentration of CO2 (SE［CO2］) promotes photosynthesis, but the promotion of elevated ［CO2］ (E ［CO2］) to photosynthesis might disappear after long term treatment (so called “CO2 acclimation”); this might result from the associated inhibition of nitrate assimilation. The present study investigated the physiological effects of short term (8 days) and long term (40 days) exposure to E［CO2］ when these were combined with different forms of inorganic N (full N; nitrate (NO3-) N) in the invasive species Eupatorium adenophorum. Exposure to E［CO2］ increased the biomass of Eadenophorum, regardless of the duration of exposure to E［CO2］ and the type of inorganic N that was supplied. E［CO2］ could promote the photosynthesis of Eadenophorum seedlings fertilised with non depleted Hoagland solutions (full N). For plants fertilised with NH4+ depleted Hoagland solution (NO3- N), LE［CO2］ treatment promoted the photosynthesis of Eadenop horum, but the promotion of photosynthesis by E［CO2］ disappeared under SE［CO2］ conditions. Photosynthetic pigments contents were determined to estimate potential changes in the photosynthetic capacity of Eadenophorum. For plants fertilised with non depleted Hoagland solution, there were no significant differences in chlorophyll among the three ［CO2］ treatments, but the treatment of SE［CO2］ increased the levels of chlorophyll in leaves. The apparent promotion of biomass accumulation and photosynthesis at LE［CO2］ without a decrease in chlorophyll indicates that Eadenophorum might not acclimate to long term exposure to E［CO2］. NH4+ depletion did not affect the capacity of LE［CO2］ to promote the photosynthesis of Eadenophorum. Thus, considering some plants fertilised with NO3- acclimating to LE［CO2］, Eadenophorum might be more competitive in areas where the soils are relatively poor in NH4+ as levels of atmospheric CO2 continue to rise.     

Ca2+ dynamics in a pollen grain and papilla cell during pollination of Arabidopsis

Ca2+ dynamics in the growing pollen tube have been well documented in vitro using germination assays and Ca2+ imaging techniques. However, very few in vivo studies of Ca2+ in the pollen grain and papilla cell during pollination have been performed. We expressed yellow cameleon, a Ca2+ indicator based on green fluorescent protein, in the pollen grains and papilla cells of Arabidopsis (Arabidopsis thaliana) and monitored Ca2+ dynamics during pollination. In the pollen grain, [Ca2+]cyt increased at the potential germination site soon after hydration and remained augmented until germination. As in previous in vitro germination studies, [Ca2+]cyt oscillations were observed in the tip region of the growing pollen tube, but the oscillation frequency was faster and [Ca2+]cyt was higher than had been observed in vitro. In the pollinated papilla cell, remarkable increases in [Ca2+]cyt occurred three times in succession, just under the site of pollen-grain attachment. [Ca2+]cyt increased first soon after pollen hydration, with a second increase occurring after pollen protrusion. The third and most remarkable [Ca2+]cyt increase took place when the pollen tube penetrated into the papilla cell wall.     

开花期低温胁迫对水稻花粉性状及剑叶理化特性的影响

以耐冷水稻品种996和冷敏感品种4628为材料,开花期在人工气候室进行7 d低温（06:00-8:00和19:00-23:00,19 ℃;08:00-10:00和16:00-19:00,21 ℃;10:00-16:00,23 ℃;23:00-06:00,17 ℃）和适温（06:00-8:00和19:00-23:00,24 ℃;08:00-10:00和16:00-19:00,26 ℃;10:00-16:00,30 ℃;23:00-06:00,22 ℃)处理,研究了低温胁迫对水稻花药开裂、花粉性状及剑叶叶绿素、可溶性糖和可溶性蛋白质含量、膜透性等理化特性的影响.结果表明：开花期低温胁迫导致水稻花药开裂系数、花粉萌发率显著下降,中部和下部颖花的不育花粉率增加.996的花药开裂系数和花粉萌发率显著高于4628,表明耐冷品种996在开花期低温胁迫下能保持较好的花粉散落特性和花粉萌发特性.低温胁迫下,996的剑叶可溶性蛋白质和游离脯氨酸含量及其增幅显著高于4628,而丙二醛含量和相对电导率及其增幅却显著低于4628,说明耐冷品种在低温胁迫下的保护性反应更迅速和强烈,其膜结构及功能更稳定.     

布氏田鼠冷暴露中褐色脂肪组织的增补及解偶联蛋白基因表达

布氏田鼠 (Microtusbrandti)随机分组暴露在冷环境 [12L∶12D ,(4± 2 )℃ ]中 12h ,1d ,3d ,7d ,14d ,2 1d和 2 8d ;对照组生活在温暖环境下 [12L∶12D ,(2 5± 2 )℃ ]。与对照组相比 ,布氏田鼠的褐色脂肪组织 (BAT)重量在冷暴露 12h～ 3d时降低 ,7～ 2 1d时则增加。 7～ 2 8d冷暴露组动物的BAT总蛋白和总DNA含量均比对照组明显提高。冷环境中的布氏田鼠解偶联蛋白 (UCP)的mRNA随时间的延长而表达上调 ,在冷暴露 2 1d时达到高峰。结果表明 ,冷暴露能够诱导布氏田鼠BAT细胞增补和UCP基因表达 ,从而使适应性产热增加。     

Osmo-sensitive and stretch-activated calcium-permeable channels in Vicia faba guard cells are regulated by actin dynamics

In responses to a number of environmental stimuli, changes of cytoplasmic [Ca(2+)](cyt) in stomatal guard cells play important roles in regulation of stomatal movements. In this study, the osmo-sensitive and stretch-activated (SA) Ca(2+) channels in the plasma membrane of Vicia faba guard cells are identified, and their regulation by osmotic changes and actin dynamics are characterized. The identified Ca(2+) channels were activated under hypotonic conditions at both whole-cell and single-channel levels. The channels were also activated by a stretch force directly applied to the membrane patches. The channel-mediated inward currents observed under hypotonic conditions or in the presence of a stretch force were blocked by the Ca(2+) channel inhibitor Gd(3+). Disruption of actin filaments activated SA Ca(2+) channels, whereas stabilization of actin filaments blocked the channel activation induced by stretch or hypotonic treatment, indicating that actin dynamics may mediate the stretch activation of these channels. In addition, [Ca(2+)](cyt) imaging demonstrated that both the hypotonic treatment and disruption of actin filaments induced significant Ca(2+) elevation in guard cell protoplasts, which is consistent with our electrophysiological results. It is concluded that stomatal guard cells may utilize SA Ca(2+) channels as osmo sensors, by which swelling of guard cells causes elevation of [Ca(2+)](cyt) and consequently inhibits overswelling of guard cells. This SA Ca(2+) channel-mediated negative feedback mechanism may coordinate with previously hypothesized positive feedback mechanisms and regulate stomatal movement in response to environmental changes.     

Modulation of reactive oxygen species production during osmotic stress in Arabidopsis thaliana cultured cells: involvement of the plasma membrane Ca2+-ATPase and H+-ATPase

In Arabidopsis thaliana cells, hypoosmotic treatment initially stimulates Ca2+ influx and inhibits its efflux and, concurrently, promotes a large H2O2 accumulation in the external medium, representative of reactive oxygen species (ROS) production. After the first 10-15 min, Ca2+ influx rate is, however, lowered, and a large rise in Ca2+ efflux, concomitant with a rapid decline in H2O2 level, takes place. The drop of the H2O2 peak, as well as the efflux of Ca2+, are prevented by treatment with submicromolar concentrations of eosin yellow (EY), selectively inhibiting the Ca2+-ATPase of the plasma membrane (PM). Comparable changes of Ca2+ fluxes are also induced by hyperosmotic treatment. However, in this case, the H2O2 level does not rise, but declines below control levels when Ca2+ efflux is activated. Also K+ and H+ net fluxes across the PM and cytoplasmic pH (pH(cyt)) are very differently influenced by the two opposite stresses: strongly decreased by hypoosmotic stress and increased under hyperosmotic treatment. The H2O2 accumulation kinetics, followed as a function of the pH(cyt) changes imposed by modulation of the PM H+-ATPase activity or weak acid treatment, show a close correlation between pH(cyt) and H2O2 formed, a larger amount being produced for changes towards acidic pH values. Overall, these results confirm a relevant role for the PM Ca2+-ATPase in switching off the signal triggering ROS production, and propose a role for the PM H+-ATPase in modulating the development of the oxidative wave through the pH(cyt) changes following the changes of its activity induced by stress conditions.     

开放式O3浓度增高对小麦籽粒蛋白的影响

于2006—2009年应用FACE研究平台,设计O₃浓度增高50%(E)和自然浓度(CK)两个处理,采用烟农19、扬麦16、扬麦15和扬辐麦2号4个品种,研究了O₃浓度增高对不同类型小麦品种籽粒蛋白质组分及其动态的影响.结果表明：随着O3浓度增高,籽粒蛋白质含量上升,3个年度上升幅度分别为7.55%～16.37%（2006—2007年度）、4.93%～22.63%（2007—2008年度）和2.29%～17.65%（2008—2009年度）,处理间、品种和年度间差异显著;但籽粒蛋白质产量显著或极显著下降,3个年度降幅分别为1.83%～11.64%（2006—2007年度）, -0.41%～24.22%（2007—2008年度）和-1.90%～15.81%（2008—2009年度）.籽粒蛋白质4种组分（清蛋白、球蛋白、醇溶蛋白和谷蛋白）含量均表现为O₃浓度增高处理高于自然浓度处理,品种间和年度间差异显著,且处理间醇溶蛋白和球蛋白含量差异显著,而清蛋白和谷蛋白含量差异不显著.     

Arabidopsis thaliana root non-selective cation channels mediate calcium uptake and are involved in growth

Calcium is a critical structural and regulatory nutrient in plants. However, mechanisms of its uptake by root cells are poorly understood. We have found that Ca2+ influx in Arabidopsis root epidermal protoplasts is mediated by voltage-independent rapidly activating Ca2+-permeable non-selective cation channels (NSCCs). NSCCs showed the following permeability (P) sequence: PCa (1.00) = PBa (0.93) > PZn (0.51), PCa/PNa = 0.19, PCa/PK = 0.14. They were inhibited by quinine, Gd3+, La3+ and the His modifier diethylpyrocarbonate, but not by the Ca2+ or K+ channel antagonists, verapamil and tetraethylammonium (TEA+). Single channel conductance measured in 20 mm external Ca2+ was 5.9 pS. Calcium-permeable NSCCs co-existed with hyperpolarisation-activated Ca2+ channels (HACCs), which activated 40-60 min after forming the whole-cell configuration. HACCs activated at voltages <-130 to -150 mV, showed slow activation kinetics and were regulated by cytosolic Ca2+ ([Ca2+]cyt). Using aequorin-expressing plants, a linear relationship between membrane potential (Vm) and resting [Ca2+]cyt was observed, indicating the involvement of NSCCs. Intact root 45Ca2+ influx was reduced by Gd3+ (NSCC blocker) but was verapamil and TEA+ insensitive. In the root elongation zone, both root net Ca2+ influx (measured by Ca2+-selective vibrating microelectrode) and NSCC activity were increased compared to the mature epidermis, suggesting the involvement of NSCC in growth. A Ca2+ acquisition system based on NSCC and HACC co-existence is proposed. In mature epidermal cells, NSCC-mediated Ca2+ influx dominates whereas in specialised root cells (root hairs and elongation zone cells) where elevated [Ca2+]cyt activates HACCs, HACC-mediated Ca2+ influx predominates.     

Protein kinase C stimulates dense tubular Ca2+ uptake in the intact human platelet by increasing the Vm of the Ca(2+)-ATPase pump: stimulation by phorbol ester, inhibition by calphostin C.

The effects of protein kinase C (PKC) on Ca2+ transport were investigated in human intact platelets. The indicator quin2 was used to measure the free cytoplasmic Ca2+ concentration ([Ca2+]cyt) and to search for possible PKC effects on the Ca(2+)-ATPase extrusion pump located in the plasma membrane. The Ca2+ indicator chlorotetracycline (CTC) was used to study PKC effects on the dense tubular Ca(2+)-ATPase uptake pump. The activity of PKC was stimulated by phorbol 12-myristate 13-acetate (PMA) and was inhibited with calphostin C. Neither PKC activation nor inhibition had any effect on [Ca2+]cyt or the Ca2+ extrusion pump. Substantial activation of the dense tubular pump was observed with PMA. In resting platelets bathed in 2 mM external Ca2+ giving [Ca2+]cyt = 102-106 nM, activation of PKC by PMA (100 nM) increases the rate and extent of dense tubular Ca2+ uptake to 1.62 +/- 0.35 and 1.25 +/- 0.3 times control value (respectively). The Vm of the dense tubular pump was measured by using ionomycin to manipulate [Ca2+]cyt. It is shown that PMA increases the Vm by a factor of 1.7 +/- 0.4 but has no effect on the Km value (= 180 nM). An unexpected finding was that PKC activity supports a portion of the basal activity of the dense tubular Ca2+ pump in resting platelets. Preincubation with the inhibitor calphostin C (100 nM) decreases the rate and extent of dense tubular Ca2+ uptake in resting platelets by 38 +/- 5% and 29 +/- 21% (respectively). This is due to a 28 +/- 9% decrease in the Vm of the dense tubular pump. This suggests that there is a low level of stimulation of dense tubular Ca2+ pump mediated by PKC in resting platelets.     

长春花中定向诱导长春碱生物半合成方法的研究

当使用诱导剂为 0.01 mol·L^-1 (Zn²⁺)+0.5 mg·L^-1 (IAA)时, 打浆时间为2.5 min、长春碱通气量为0.30 L·min^-1通气5 min、培养固液比为1：15、培养时间为2 h、培养温度为20℃~25 ℃时,生物半合成法可提高50％左右的长春碱。     

黄淮海地区不同冬春性小麦抗冻能力及冻害指标Ⅰ.隆冬期不同冬春性小麦抗冻能力比较

通过两年室外盆栽和室内人工控制试验,建立了不同冬春性小麦品种死苗率与低温的定量关系,确定了隆冬期不同冬春性小麦品种死苗率达10%、20%和50%的临界低温以及抗冻能力密度.结果表明： 强冬性小麦品种抗冻能力最强,半致死温度最低（燕大1817为-21.5 ℃,京411为-21.2 ℃）,其次是冬性品种（农大211为-21.1 ℃,农大5363为-20.3 ℃）和弱冬性品种（郑366为-18.5 ℃,平安8号为-18.4 ℃）,春性品种抗冻能力最弱（郑9023为-15.4 ℃,偃展4110为-14.7 ℃）.当温度降低到冬小麦冻害发生临界温度后,温度每降低1 ℃,弱冬性小麦（郑366和平安8号）死苗率增量最大,分别增加16.8%和25.8%,冬性小麦（农大211和农大5363）死苗率分别增加14.7%和18.9%,强冬性小麦（燕大1817和京411）死苗率分别增加15.4%和13.1%,春性小麦（郑9023和偃展4110）死苗率分别增加13.8%和15.1%,说明冻害发生后若持续降温,弱冬性品种遭受冻害风险更大.     

Heterogeneity of hypoxia-mediated decrease in I(K(V)) and increase in [Ca2+](cyt) in pulmonary artery smooth muscle cells

Hypoxic pulmonary vasoconstriction is caused by a rise in cytosolic Ca(2+) ([Ca(2+)](cyt)) in pulmonary artery smooth muscle cells (PASMC) via multiple mechanisms. PASMC consist of heterogeneous phenotypes defined by contractility, proliferation, and apoptosis as well as by differences in expression and function of various genes. In rat PASMC, hypoxia-mediated decrease in voltage-gated K(+) (Kv) currents (I(K(V))) and increase in [Ca(2+)](cyt) were not uniformly distributed in all PASMC tested. Acute hypoxia decreased I(K(V)) and increased [Ca(2+)](cyt) in approximately 46% and approximately 53% of PASMC, respectively. Using combined techniques of single-cell RT-PCR and patch clamp, we show here that mRNA expression level of Kv1.5 in hypoxia-sensitive PASMC (in which hypoxia reduced I(K(V))) was much greater than in hypoxia-insensitive cells (in which hypoxia negligibly affected I(K(V))). These results demonstrate that 1) different PASMC express different Kv channel alpha- and beta-subunits, and 2) the sensitivity of a PASMC to acute hypoxia partially depends on the expression level of Kv1.5 channels; hypoxia reduces whole-cell I(K(V)) only in PASMC that express high level of Kv1.5. In addition, the acute hypoxia-mediated changes in [Ca(2+)](cyt) also vary in different PASMC. Hypoxia increases [Ca(2+)](cyt) only in 34% of cells tested, and the different sensitivity of [Ca(2+)](cyt) to hypoxia was not related to the resting [Ca(2+)](cyt). An intrinsic mechanism within each individual cell may be involved in the heterogeneity of hypoxia-mediated effect on [Ca(2+)](cyt) in PASMC. These data suggest that the heterogeneity of PASMC may partially be related to different expression levels and functional sensitivity of Kv channels to hypoxia and to differences in intrinsic mechanisms involved in regulating [Ca(2+)](cyt).     

Comparison of Properties of the Plasma Membrane Ca2+ -ATPase from Wheat Root and Leaf

The properties of plasma membrane Ca2 + -ATPases from wheat ( Triticum aestivum L. cv. Lengchun No. 13) root and leaf were compared, and their different properties were analyzed in association with the differentia of the functions of these two organs and their relevant environments. Root plasma membrane Ca2 + -ATPase showed a high activity in a broad range of pH and an optimum reaction temperature of 45 ℃, while the leaf enzyme activated in a narrow range of pH and an optimum reaction temperature of 50 ℃. Hill coefficient of root plasma membrane Ca2 + -ATPase for ATP was 1.6, revealing an obvious positive cooperativity. In contrast, that of leaf plasma membrane Ca2 +-ATPase was 1.0, being in keeping with Michaelis-Menten dynamics. For Ca2 + activation, Hill coefficient of plasma membrane Ca2 + -ATPases from both organs were less than 1, suggesting that both had negative cooperativity. The enzymes were activated by calmodulin and inhibited by Mg2+.     

小麦叶肉细胞原生质体中微管骨架的排列格局与[Ca^2＋]cyt的关系

以小麦叶肉细胞原生质体为材料,通过免疫荧光标记和Ca^2＋荧光染料的装载并结合药物学试验,借助激光共聚焦扫描显微镜观察,探讨微管骨架和Ca^2＋之间的内在联系。试验结果表明,[Ca^2＋]cyt的升高能够诱发微管骨架的解聚;而微管骨架的解聚也会促使胞外Ca^2＋内流,进而造成[Ca^2＋]cyt的升高。