小麦叶肉细胞原生质体中微管骨架的排列格局与[Ca2+]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。     

激发子诱发小麦叶肉细胞原生质体微管骨架排列格局与[Ca2+]cyt的变化

以小麦叶肉细胞原生质体-激发子互作为研究体系,通过免疫荧光标记和Ca~(2 )荧光染料的装载并结合药理学试验,借助激光共聚焦扫描显微镜观察,探讨小麦抵抗叶锈菌侵染过程中微管骨架和Ca~(2 )之间的内在联系。试验结果表明,激发子处理可引起抗性品种原生质体[Ca~(2 )]_(cyt)的升高并诱发微管骨架的解聚,预解聚微管骨架,再用激发子处理,可使抗性品种原生质体[Ca~(2 )]_(cyt)的升高幅度增加。     

Ca^2＋参与茉莉酸诱导蚕豆气孔关闭的信号转导

以Fluo-3 AM为Ca^2＋荧光探针,结合激光共聚焦扫描显微技术,观察到在处理后数十秒内,气孔关闭之前,茉莉酸（JA）可引起[Ca^2＋]cyt的迅速上升;对照和JA的前体物亚麻酸（LA）几乎不能引起[Ca^2＋]cyt的明显变化;钙的螯合剂EGTA预处理可完全阻断JA诱导气孔关闭的效应,并且JA不再引起保卫细胞[Ca^2＋]cyt增加;质膜Cah通道的抑制剂硝苯吡啶（nifedipine,NIF）可减弱JA诱导气孔关闭的效应,也使JA诱导保卫细胞[Ca^2＋]cyt增加的幅度有所下降;胞内Ca^2＋释放的抑制剂钌红不能明显改变JA诱导气孔关闲的趋势,但使JA引起的保卫细胞[Ca^2＋]cyt增加有所降低。实验结果表明：Ca^2＋参与JA诱导气孔关闭的信号转导;推测JA引起的[Ca^2＋]cyt升高可能主要来源于胞外,但不能完全排除胞内Ca^2＋的释放。     

低温处理对冬、春小麦细胞Ca^2＋时空变化的影响

用Fluo-3/AM染色,通过激光扫描共聚焦显微镜（LSCM）方法,对静息态及连续降温条件下不同抗寒性小麦（Triticum aestivum L.）叶肉细胞原生质体[Ca^2 ]cyt（the free Ca^2 concentration in the cytoplasm）的时空变化进行了比较。结果表明,静息态下小麦原生质体整体荧光强度基本不变,暗示[Ca^2 ]cyt能维持在一稳定水平;同时,不同品种小麦间也显示了[Ca^2 ]cyt水平荧光强度的不同。温度由15℃连续降至约2℃时,抗寒冬小麦[Ca^2 ]cyt出现升高后的回复,2℃之后逐渐升高;冷敏感春小麦则无此回复过程,而是一直升高到最大值。推测这一不同的动态变化最终决定了植物在低温下产生冷适应的不同能力。这进一步为“Ca^2 是低温下生理信号的传导”这一假说提供了新的证据。     

保卫细胞钙信号的研究进展

钙(Ca^2 )是多种信号途径的第二信使。Ca^2 成像技术的成熟和发展为显示保卫细胞胞质Ca^2 浓度([Ca^2 ]cyt)的分布及外界刺激引起[Ca^2 ]cyt的变化模式提供了很好的研究工具,关于细胞内外Ca^2 库释放Ca^2 的机制也有了较清楚的认识。拟南芥突变体的研究使Ca^2 信号上游分子及其排序更加明确,[Ca^2 ]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。     

Water Channels Are Involved in Stomatal Oscillations Encoded by Parameter-Specific Cytosolic Calcium Oscillations

Earlier studies have shown that various stimuli can induce specific cytosolic calcium （[Ca^2＋]cyt） oscillations in guard cells and various oscillations in stomatal apertures. Exactly how [Ca^2＋]cyt oscillation signaling functions in stomatal oscillation is not known. In the present study, the epidermis of broad bean （Vicia faba L.） was used and a rapid ion-exchange treatment with two shifting buffers differing in K^＋ and Ca^2＋ concentrations was applied. The treatment for fivetransients at a 10-min transient period induced clear and regular stomatal oscillation. However, for other transient numbers and periods, the treatments induced some Irregular oscillations or even no obvious oscillations in stomatal aperture. The results indicate that stomatal oscillation Is encoded by parameter-specific [Ca^2＋]cyt oscillation： the parameters of [Ca^2＋]cyt oscillation affected the occurrence rate and the parameters of stomatal oscillation. The water channel inhibitor HgCl2 completely Inhibited stomatal oscillation and the inhibitory effect could be partially reversed by β-mercaptoethanol （an agent capable of reversing water channel inhibition by HgCl2）. Other Inhibitory treatments against Ion transport （i.e. the application of LaCIs, EGTA, or tetraethylammonlum chloride （TEACI）） weakly impaired stomatal oscillation when the compounds were added after rapid ion-exchange treatment. If these compounds were added before rapid-ion exchange treatment, the inhibitory effect was much more apparent （except In the case of TEACI）. The results of the present study suggest that water channels are involved In stomatal oscillation as a downstream element of [Ca^2＋]cyt oscillation signaling.     

Actin Dynamics Regulates Voltage-Dependent Calcium-Permeable Channels of the Vicia faba Guard Cell Plasma Membrane

Free cytosolic Ca^2＋ （[Ca^2＋]cyt） is an ubiquitous second messenger in plant cell signaling, and [Ca^2＋]cyt elevation is associated with Ca^2＋-permeable channels in the plasma membrane and endomembranes regulated by a wide range of stimuli. However, knowledge regarding Ca^2＋ channels and their regulation remains limited in planta. A type of voltage- dependent Ca^2＋-permeable channel was identified and characterized for the Vicia faba L. guard cell plasma membrane by using patch-clamp techniques. These channels are permeable to both Ba^2＋ and Ca^2＋, and their activities can be inhibited by micromolar Gd^3＋. The unitary conductance and the reversal potential of the channels depend on the Ca^2＋ or Ba^2＋ gradients across the plasma membrane. The inward whole-cell Ca^2＋ （Ba^2＋） current, as well as the unitary current amplitude and NPo of the single Ca^2＋ channel, increase along with the membrane hyperpolarization. Pharmacological experiments suggest that actin dynamics may serve as an upstream regulator of this type of calcium channel of the guard cell plasma membrane. Cytochalasin D, an actin polymerization blocker, activated the NPo of these channels at the single channel level and increased the current amplitude at the whole-cell level. But these channel activations and current increments could be restrained by pretreatment with an F-actin stabilizer, phalloidin. The potential physiological significance of this regulatory mechanism is also discussed.     

以单细胞免疫荧光技术观察烟草细胞融合过程中微管骨架的变化

本文建立了单细胞免疫荧光标记技术并以此结合单对细胞融合技术对细胞融合过程中微管骨架组织形式的动态变化进行了追踪观察。发现在聚乙二醇（PEG）诱导条件下,一旦细胞开始粘连,细胞内微管骨架便开始解聚。在细胞融合的整个过程中一直维持着这种解聚的状态,直到融合完成,在后续的培养中微管骨架才重新出现。在微管骨架呈解聚状态时融合产物不能完成与另外的细胞融合。实验揭示了细胞的再融合能力可能受细胞本身微管骨架状态的影响。该结果为解释高等植物如何避免多精入卵提供了新的可能性。     

以单细胞免疫荧光技术观察烟草细胞融合过程中微管骨架的变化

本文建立了单细胞免疫荧光标记技术并以此结合单对细胞融合技术对细胞融合过程中微管骨架组织形式的动态变化进行了追踪观察。发现在聚乙二醇(PEG)诱导条件下,一旦细胞开始粘连,细胞内微管骨架便开始解聚。在细胞融合的整个过程中一直维持着这种解聚的状态,直到融合完成,在后续的培养中微管骨架才重新出现。在微管骨架呈解聚状态时融合产物不能完成与另外的细胞融合。实验揭示了细胞的再融合能力可能受细胞本身微管骨架状态的影响。该结果为解释高等植物如何避免多精入卵提供了新的可能性。     

有氧运动对大鼠心肌活细胞核钙转运功能的影响

目的：研究有氧运动后心肌细胞核钙动态变化的生物学机制。方法：采用激光扫描共聚焦显微术（LSCM）对STDBT和Fluo-3Ca^2＋荧光试剂负载的急性分离的有氧运动大鼠心肌活细胞内游离Ca^2＋动态变化进行研究。结果：以胞核特异性Ca^2＋荧光探针STDBT-AM标记的心室肌细胞胞体四周荧光微弱,横纹、横小管隐约可见,但在胞体核的部位荧光分布最强,出现强绿色区域,核膜边界清晰,表明STDBT通过胞浆而特异性聚集在胞核区域,具有靶向性导入胞核区域的特征。有氧训练组与安静对照组比较,[Ca^2＋]n的基值和峰值变化特征与Fluo-3相同。有氧训练组加入异丙肾上腺素后,[Ca^2＋]n显著性增加,其变化率为33.3412%（P〈0.01〉。异丙肾上腺素作用前、后大鼠心肌细胞[Ca^2＋]n发生较明显的变化。[Ca^2＋]n有氧运动组加药后较加药前显著性增加,其变化率为33.224%。结论：本文首次研究了有氧运动训练条件下心肌活细胞核Ca^2＋的变化,证实了有氧运动训练可显著增加心肌细胞[Ca^2＋]n变化水平,并首次初步探讨了有氧运动训练条件下心肌细胞核Ca^2＋的转运功能,异丙肾上腺素可显著升高[Ca^2＋]n而降低[Ca^2＋]i。心肌细胞核也是心肌细胞内钙库之一,有氧训练可影响心肌细胞[Ca^2＋]n调控。     

水稻 Ca2+/H+ 反向转运体 OsCAX3 的功能分析和亚细胞定位研究

Ca2+/H+ 反向转运体作为一类 Ca2+外向转运器,在植物的营养和信号转导中起着非常重要的作用 . 克隆了水稻 Ca2+/H+ 反向转运体基因 OsCAX3 ,序列分析表明 OsCAX3 具有 11 个跨膜区,其中在第 6 和第 7 个跨膜区之间有一个 17 个氨基酸组成的酸性基序 (acid motif) ,功能互补实验证明 OsCAX3 具有转运 Ca2+ 的功能,并且其 N 端 26 个氨基酸序列对转运 Ca2+ 具有一定的抑制作用 . RT-PCR 分析表明 OsCAX3 的表达受到外源 Ca2+ 的诱导 . 利用 PSORT prediction 进行亚细胞定位分析,和利用 OsCAX3-GFP 融合蛋白瞬时表达分析证明, OsCAX3 定位于细胞质膜 . 以上结果表明, OsCAX3 是一种定位于细胞质膜上的 Ca2+/H+ 反向转运体 .     

Effect of modification of cell calcium status on lectin activity

Effects of oryzalin (10 microM), an inhibitor of microtubule polymerization, on the activity of soluble and cell wall lectins were studied in 7 day-old seedlings of unhardened (23 degrees C) and cold acclimated (7 days at 2-3 degrees C) winter wheat (Triticum aestivum L.). Seedlings were grown in the presence of 25 microM and 1 mM Ca2+, 500 microM verapamil, 250 microM chlorpromazine or without modifiers of calcium status in the medium. Inhibitor of the microtubule polymerization inhibitor, likely as inhibitors of Ca(2+)-signal, decreased the activity of soluble lectins and increased that of cell wall lectins. Apparently, injury of microtubule phosphorylation results in a more considerable microtubule disorganization, than that observed after oryzalin effect. A low Ca2+ concentration (25 microM) depressed, while a high concentration (1 mM) prompted microtubule sensibility to oryzalin. Such an effect of high Ca2+ concentration may be related to destabilizative action of Ca(2+)-calmodulin in these conditions, because chlorpromazine decreased oryzalin-induced increase in the activity of cell wall lectins with 1 mM Ca2+. It is concluded that the activity of cell wall lectins depends on the microtubule status that is regulated by calcium signal.     

The Light-Dependent Transduction Pathway Controlling the Regulatory Phosphorylation of C4 Phosphoenolpyruvate Carboxylase in Protoplasts from Digitaria sanguinalis

Phosphoenolpyruvate carboxylase (PEPC) was characterized in extracts from C4 mesophyll protoplasts isolated from Digitaria sanguinalis leaves and shown to display the structural, functional, and regulatory properties typical of a C4 PEPC. In situ increases in the apparent phosphorylation state of the enzyme and the activity of its Ca2+-independent protein-serine kinase were induced by light plus NH4Cl or methylamine. The photosynthesis-related metabolite 3-phosphoglycerate (3-PGA) was used as a substitute for the weak base in these experiments. The early effects of light plus the weak base or 3-PGA treatment were alkalinization of protoplast cytosolic pH, shown by fluorescence cytometry, and calcium mobilization from vacuoles, as suggested by the use of the calcium channel blockers TMB-8 and verapamil. The increases in PEPC kinase activity and the apparent phosphorylation state of PEPC also were blocked in situ by the electron transport and ATP synthesis inhibitors DCMU and gramicidin, respectively, the calcium/calmodulin antagonists W7, W5, and compound 48/80, and the cytosolic protein synthesis inhibitor cycloheximide. These results suggest that the production of ATP and/or NADPH by the illuminated mesophyll chloroplast is required for the activation of the transduction pathway, which presumably includes an upstream Ca2+-dependent protein kinase and a cytosolic protein synthesis event. The collective data support the view that the C4 PEPC light transduction pathway is contained entirely within the mesophyll cell and imply cross-talk between the mesophyll and bundle sheath cells in the form of the photosynthetic metabolite 3-PGA.     

增强UV-B辐射对小麦叶肉细胞原生质体微丝骨架的影响

以增强UV-B（10.08 kJ/m2.d）辐射后的小麦幼苗叶肉细胞原生质体为材料,异硫氰酸荧光素标记的鬼笔环肽（FITC-Ph）为探针,利用激光共聚焦扫描显微镜,观察分析小麦叶肉细胞原生质体中微丝骨架的分布及形态变化。结果表明对照组中,叶肉细胞原生质体微丝呈现网状或平行状随机排列,形态上表现为纤维状结构。增强UV-B辐射处理后,原生质体中微丝的密集分布遭到破坏,纤丝状微丝消失,聚集成束,或呈点状、碎片状分布。     

Processes modulating calcium distribution in citrus leaves. An investigation using x-ray microanalysis with strontium as a tracer

Citrus leaves accumulate large amounts of calcium that must be compartmented effectively to prevent stomatal closure by extracellular Ca2+ and interference with Ca(2+)-based cell signaling pathways. Using x-ray microanalysis, the distribution of calcium between vacuoles in different cell types of leaves of rough lemon (Citrus jambhiri Lush.) was investigated. Calcium was accumulated principally in palisade, spongy mesophyll, and crystal-containing idioblast cells. It was low in epidermal and bundle sheath cells. Potassium showed the reverse distribution. Rubidium and strontium were used as tracers to examine the pathways by which potassium and calcium reached these cells. Comparisons of strontium and calcium distribution indicated that strontium is a good tracer for calcium, but rubidium did not mirror the potassium distribution pattern. The amount of strontium accumulated was highest in palisade cells, lowest in bundle sheath and epidermal cells, and intermediate in the spongy mesophyll. Accumulation of strontium in palisade and spongy mesophyll was accompanied by loss of potassium from these cells and its accumulation in the bundle sheath. Strontium moved apoplastically from the xylem to all cell types, and manipulation of water loss from the adaxial leaf surface suggested that diffusion is responsible for strontium movement to this side of the leaf. The results highlight the importance of palisade and spongy mesophyll as repositories for calcium and suggest that calcium distribution between different cell types is the result of differential rates of uptake. This tracer technique can provide important information about the ion uptake and accumulation properties of cells in intact leaves.     

外源胆固醇对离体原生质体在冰冻—化冻过程中的保护作用

在适宜浓度(0.1～0.4m mol/L)的胆固醇存在下,经冰冻处理的小麦叶肉原生质体在化冻后存活率均高于对照,其中以0.4m mol/L胆固醇对原生质体的保护效果最佳。胆固醇对卷心菜叶肉原生质体的抗冰冻能力具有类似效果。从而表明外源胆固醇与质膜结合后,对稳定质膜结构和保持正常功能具有一定的作用。     

增强UV-B辐射和He-Ne激光对小麦原生质体微管骨架的影响

以小麦叶片原生质体为材料,采用间接免疫荧光定位法标记其微管系统,并利用激光共聚焦扫描显微系统进行观察。研究了低剂量He-Ne激光(5mW.mm-2)、增强UV-B辐射(10.08kJ.m-2.d-1)及二者的复合处理对小麦幼苗叶肉细胞中微管骨架的影响。结果表明,增强UV-B辐射后,小麦叶片细胞中微管骨架发生解聚,呈短棒状或点状分布,微管束弥散且荧光强度减弱;而增强UV-B辐射后再施以He-Ne激光处理,小麦叶肉细胞微管骨架有部分断裂,但较单独UV-B处理组的损伤程度轻,说明低剂量的He-Ne激光可以部分修复增强UV-B辐射对微管骨架的损伤,且对微管的聚合有促进作用。     

增强UV-B辐照对小麦幼苗叶肉细胞内Ca2＋水平的研究

以小麦（Triticum aestivum）幼苗叶片为材料,利用提取原生质体方法在小麦幼苗叶肉细胞中成功地装载了钙离子荧光指示剂fluo-3/AM,采用激光共聚焦显微技术检测了增强UV-B辐射后小麦幼苗叶肉细胞内游离钙离子荧光强度的分布,并对[Ca2＋];进行了测定.结果显示,对照组细胞内钙离子荧光分布较均匀,主要分布于紧贴质膜处和核周围,UV-B辐射组钙离子荧光与对照组分布相似,但其原生质体表面不如对照组平滑;同时发现增强UV-B辐射组细胞内钙离子荧光强度值较对照组高,说明增强UV-B辐射组小麦幼苗叶肉细胞维持较高浓度的钙离子水平.这些变化表明Ca2＋信号有可能以一定的方式参与了小麦响应UV-B辐射胁迫的过程.