拟南芥钙调素定点突变基因分离及其在钙不依赖钙调素结合蛋白检测中的应用

动植物系统研究表明,钙调素不仅在结合钙离子时调节多种靶酶或靶蛋白的活性,而且没有钙离子结合时,还可以通过结合钙不依赖的钙调素结合蛋白,发挥多种生物学作用．然而,目前却没有体内分析钙调素与钙不依赖钙调素结合蛋白相互作用的方法．首先,采用定点突变的方式,得到了拟南芥钙调素亚型2的多个突变基因mCaM2,随后,大肠杆菌重组表达突变蛋白的电泳迁移率及⁴⁵Ca²⁺覆盖分析表明,得到了编码失去钙结合能力的钙调素的突变基因mCaM2₁₂₃₄, mCaM2₁₂₃₄突变钙调素中所有4个钙结合EF-hand结构域中的关键氨基酸谷氨酸均突变为谷氨酰胺．在酵母双杂交体系中,作为诱饵蛋白的突变钙调素mCaM2₁₂₃₄与我们前期体外方法报道的钙不依赖性钙调素结合蛋白AtIQD26存在相互作用．这将为钙不依赖性钙调素结合蛋白提供有用的体内研究工具,有利于我们全面认识钙-钙调素-钙调素结合蛋白信号途径．     

钙调素的结构生物学研究进展

介绍了Apo-CaM、Ca²⁺-CaM以及CaM与其靶肽及拮抗剂复合体的空间结构.钙调素(calmodulin, CaM)作为细胞多功能的Ca²⁺受体,在细胞信号转导过程中发挥重要作用.近几年对它的空间结构有了较清楚的了解,使人们能够更明确地认识CaM的Ca²⁺激活及CaM与其靶酶的作用机制.     

钙依赖的磷脂结合蛋白——钙结合蛋白中的一个新家族

钙依赖的磷脂结合蛋白是70年代末发现的一类新的钙结合蛋白,它们不同于钙调素等具有“EF”手结构的钙结合蛋白,其特点是它们与Ca²⁺结合后可以进一步与膜磷脂结合。这类蛋白质广泛存在于动物细胞,常常与质膜或内膜系统相联系。免疫化学证据和对其氨基酸顺序、cDNA序列分析表明,这是钙结合蛋白中一个包括多个成员、结构与功能相关的新家族。     

35S标记重组植物钙调素的制备方法

利用³⁵S标记的氨基酸混合物喂养工程菌,成功地制备了³⁵S标记的拟南芥钙调素亚型2（³⁵S-ACaM2）,对其纯度、放射活度、电泳行为及其灵敏性等进行了检测.结果表明从工程菌中制备的³⁵S-ACaM2纯度高、放射活度高、Ca²⁺与EGTA存在时的电泳行为与未标记的ACaM2相同,可作为一种高灵敏性的探针用于检测钙调素结合蛋白.     

豚鼠精子质膜Ca2+ -ATPase活性与精子顶体反应关系的研究

用生化测定法首次证实豚鼠精子质膜Ca²⁺-ATPase活性在精子获能和顶体反应过程中显著下降.Ca²⁺-ATPase抑制剂利尿酸(ethacrynic acid)抑制质膜Ca²⁺-ATPase活性,但钙调素(50μg/mL)的拮抗剂三氟拉嗪(TFP,200～500μmol/L)对该酶活性没有影响,说明钙调素不直接参与精子依赖于ATP的Ca²⁺的主动泵出.但钙调素与精子的Ca²⁺内流有关,钙调素拮抗剂TFP显著促进精子顶体反应和精子对Ca²⁺的摄入.Ca²⁺-ATPase抑制剂栎皮酮(quercetin)、原钒酸钠(sodiumorthovandate)、利尿磺胺(furosemide)和利尿酸均显著促进豚鼠精子的顶体反应,但却抑制精子对Ca²⁺的摄入,这无法用它们对质膜Ca²⁺-ATPase活性的抑制作用解释.推测这可能是由于Ca²⁺-ATPase抑制剂在抑制质膜Ca²⁺-ATPase活性的同时也抑制了顶体外膜或线粒体外膜上的该酶的活性,导致Ca²⁺在细胞质内的积累,进而通过负反馈机制抑制Ca²⁺进一步内流所致.另外,Ca²⁺-ATPase抑制剂对糖酵解的抑制作用也可能是Ca²⁺在细胞质中积累和抑制精子Ca²⁺摄入的原因.     

钙信号途径参与小斑病菌致病过程的调控*

为确定Ca²⁺信号途径与玉米小斑病菌致病过程的相关性,用可从不同位点阻断Ca²⁺信号转导途径的抑制剂分别处理小斑病菌的分生孢子,结果表明:Ca²⁺螯合剂EGTA、Ca²⁺通道抑制剂Verapam il、影响钙调素与钙调素依赖蛋白激酶作用位点的抑制剂KN-93,随着浓度的增加,对孢子萌发和附着胞形成过程的抑制作用明显增强;同一浓度下,抑制剂对附着胞形成过程的抑制作用大于孢子萌发过程;抑制剂可使附着胞形态明显变小甚至不能形     

钙调素及其结合蛋白BP-10对类囊体膜蛋白磷酸化的作用

报道光诱导的内源类囊体膜蛋白的磷酸化可被一种新的植物钙调素(Calmodulin ,CaM )结合蛋白BP 1 0 (CaMBP -1 0 )显著抑制 ,并且抑制作用能被外加CaM消除 .同时 ,此磷酸化反应也可被EGTA和CaM拮抗剂TFP(trifluoperazine)及W 7(N ( 6 aminohexyl) -5- chloro -1 naphthalenesulfonamide)抑制 .提示 :( 1 )Ca ²⁺和CaM可能参与并调节植物光合作用 ;( 2 )催化类囊体膜蛋白磷酸化的激酶可能受Ca ²⁺和CaM调控 .进一步实验表明BP -1 0对类囊体膜蛋白的脱磷酸化作用无任何影响 .     

钙调神经磷酸酶的研究进展

钙调神经磷酸酶(CaN)是一种受Ca²⁺/钙调素调节的丝/苏氨酸蛋白磷酸酶,广泛存在于哺乳动物的组织细胞中,作为Ca²⁺信号下游的一种效应分子,参与多种细胞功能的调节.在T细胞活化的信号传导中起到调节枢纽的作用;在神经递质的释放、突触可塑性方面亦有重要的调节作用.新近的研究表明,CaN在心肌肥厚的发生发展中起到中心作用.对CaN的分子结构、酶学特性、组织分布、信号传导及生物学功能方面的研究进展进行了介绍.     

Ca2+信号途径参与稻瘟病菌分生孢子萌发及附着胞形成的调控

为了确定Ca²⁺信号途径是否参与、在哪一时期参与稻瘟病菌分生孢子萌发及附着胞形成过程的调控,用四种可从不同位点阻断Ca²⁺信号途径的抑制剂分别处理分生孢子,观察抑制剂对孢子萌发及附着胞形成过程的抑制作用。结果表明:Ca²⁺螯合剂EGTA、Ca²⁺通道抑制剂Verapamil、抑制磷脂酶C活性的抑制剂U-73122、影响钙调素与钙调素依赖蛋白激酶作用位点的抑制剂KN-93,随着浓度的增加,对孢子萌发和附着胞形成过程的抑制作用明显增强;同一浓度下,抑制剂对附着胞形成过程的抑制作用大于孢子萌发过程;抑制剂影响孢子萌发和附着胞形成过程在萌发早期(1~4h)最有效;在完全被抑制、不能萌发的孢子内出现了许多颗粒状囊泡;抑制剂可使附着胞形态明显变小甚至不能形成。以上结果表明钙信号途径参与了稻瘟病菌孢子萌发及疏水条件下附着胞形成过程的调控。     

胞外ATP对AlCl3诱导的细胞死亡过程中胞内H2O2和Ca2+水平的影响及其生理机制分析

该实验以烟草悬浮细胞 BY 2 为材料,在烟草悬浮细胞中分别加入0.05、0.10、0.15、0.20 mmol·L^-1AlCl₃,以等体积去离子水处理的悬浮细胞液为对照,并依据前述实验结果选择0.15 mmol·L^-1 AlCl₃,分别添加5 mmol·L^-1 DMTU(H₂O₂ 抑制剂)、20 μmol·L^-1CaCl₂、15 μmol·L^-1 LaCl₃(Ca²⁺通道抑制剂)和50 μmol·L^-1 ATP设计多项处理,分析胞外ATP(eATP)对铝离子(Al³⁺)胁迫引起的植物细胞死亡及其胞内H₂O₂、Ca²⁺的影响,以揭示Al³⁺胁迫下植物调节细胞死亡的可能机制,进一步扩展对eATP功能的认知。结果显示：(1)随着 AlCl₃ 胁迫浓度的提高,细胞死亡水平和胞内H₂O₂水平上升,而胞内Ca²⁺和eATP水平则逐渐降低。(2)外援施加H₂O₂抑制剂 DMTU(二甲基硫脲)和Ca²⁺能够有效缓解AlCl₃诱导的细胞死亡水平的上升;而Ca²⁺通道抑制剂LaCl₃(三氯化镧)则加剧了AlCl₃胁迫下的细胞死亡。(3)在AlCl₃胁迫下对细胞添加外源ATP,能够缓解AlCl₃胁迫下胞内H₂O₂水平上升和Ca²⁺水平下降的同时,并显著降低AlCl₃胁迫导致的细胞死亡。研究表明, Al³⁺以剂量依赖的模式提升细胞死亡和细胞内H₂O₂的水平并降低胞内Ca²⁺和eATP水平,AlCl₃诱导的细胞死亡受到H₂O₂和Ca²⁺水平变化的调节,eATP可以通过调节H₂O₂与Ca²⁺水平缓解AlCl₃诱导的细胞死亡。推测Al³⁺胁迫可能通过抑制钙离子通道而破坏了细胞内H₂O₂和Ca²⁺之间的协同关系,外源ATP对Al³⁺诱导H₂O₂上升的缓解作用可能是由于其提升了细胞的抗氧化能力。     

植物体内钙信号及其在调节干旱胁迫中的作用

钙作为植物体内第二信使广泛参与了植物响应的各种非生物和生物胁迫的信号传导。胁迫信号通过激活位于细胞质膜上的钙离子通道,产生胞质内特异性的钙信号,传递至钙信号感受蛋白,如钙调素(calmodulin,CaM)、钙依赖蛋白激酶(Ca2+-dependent protein kinases,CDPK)和类钙调磷酸酶B蛋白(calcineurin B-like protein,CBL)等,进而引起胞内一系列生理生化变化,最终对胁迫做出响应。钙信号在植物响应干旱胁迫信号系统中起枢纽作用,主要通过调节气孔运动,水通道蛋白(aquaporin,AQP)和抗氧化酶活性来减少水分流失,提高水分利用率,最终降低干旱对植物细胞的伤害,并具有一定的生态学功能。该文对近年来国内外有关植物体内钙信号的研究进展以及在干旱逆境中的调节作用进行综述,并对今后的研究做了展望。     

Crosstalk During Fruit Ripening and Stress Response Among Abscisic Acid,Calcium-Dependent Protein Kinase and Phenylpropanoid

Phenylpropanoids are secondary metabolites produced by plants. They, by differential expression, are involved in responses to biotic and abiotic stresses and confer plant plasticity. In addition, they are synthesized under normal conditions during the fruit-ripening process. Therefore, the understanding of the mechanics involved in the accumulation of these compounds in plants is of extreme importance for the development of plants with greater resistance and tolerance to biotic and abiotic stresses, and plants with greater functional potential. There is evidence that one of the pathways of the induction of phenylpropanoids is dependent on abscisic acid (ABA) and it is generated by a signaling cascade involving calcium (Ca²⁺) and Ca²⁺-dependent protein kinases (CDPKs). Plants have several Ca²⁺ binding proteins that act as cellular sensors and represent the first points of signal transduction. CDPKs are mono-molecular Ca²⁺-sensor/kinase-effector proteins, which perceive Ca²⁺ signals and translate them into protein phosphorylation and thus represent an ideal tool for signal transduction. However, the mechanisms involved in the ABA–CDPK–phenylpropanoids crosstalk under stress conditions and during fruit ripening remains uncertain. Therefore, this review seeks to surface a new line of evidence as an attempt to understand the manner in which the induction of phenylpropanoids occurs in plants.     

Analysis of EF-hand-containing proteins in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Background  

In plants, calcium (Ca²⁺) has emerged as an important messenger mediating the action of many hormonal and environmental signals, including biotic and abiotic stresses. Many different signals raise cytosolic calcium concentration ([Ca²⁺]_cyt), which in turn is thought to regulate cellular and developmental processes via Ca²⁺-binding proteins. Three out of the four classes of Ca²⁺-binding proteins in plants contain Ca²⁺-binding EF-hand motif(s). This motif is a conserved helix-loop-helix structure that can bind a single Ca²⁺ ion. To identify all EF-hand-containing proteins in Arabidopsis, we analyzed its completed genome sequence for genes encoding EF-hand-containing proteins.     

Experimental and computational approaches for the study of calmodulin interactions

Ca²⁺, a universal messenger in eukaryotes, plays a major role in signaling pathways that control many growth and developmental processes in plants as well as their responses to various biotic and abiotic stresses. Cellular changes in Ca²⁺ in response to diverse signals are recognized by protein sensors that either have their activity modulated or that interact with other proteins and modulate their activity. Calmodulins (CaMs) and CaM-like proteins (CMLs) are Ca²⁺ sensors that have no enzymatic activity of their own but upon binding Ca²⁺ interact and modulate the activity of other proteins involved in a large number of plant processes. Protein-protein interactions play a key role in Ca²⁺/CaM-mediated in signaling pathways. In this review, using CaM as an example, we discuss various experimental approaches and computational tools to identify protein-protein interactions. During the last two decades hundreds of CaM-binding proteins in plants have been identified using a variety of approaches ranging from simple screening of expression libraries with labeled CaM to high-throughput screens using protein chips. However, the high-throughput methods have not been applied to the entire proteome of any plant system. Nevertheless, the data provided by these screens allows the development of computational tools to predict CaM-interacting proteins. Using all known binding sites of CaM, we developed a computational method that predicted over 700 high confidence CaM interactors in the Arabidopsis proteome. Most (>600) of these are not known to bind calmodulin, suggesting that there are likely many more CaM targets than previously known. Functional analyses of some of the experimentally identified Ca²⁺ sensor target proteins have uncovered their precise role in Ca²⁺-mediated processes. Further studies on identifying novel targets of CaM and CMLs and generating their interaction network - “calcium sensor interactome” - will help us in understanding how Ca²⁺ regulates a myriad of cellular and physiological processes.     

Structure and function of the CBL–CIPK Ca2+-decoding system in plant calcium signaling

Calcium is a crucial messenger in many growth and developmental processes in plants. The central mechanism governing how plant cells perceive and respond to environmental stimuli is calcium signal transduction, a process through which cellular calcium signals are recognized, decoded, and transmitted to elicit downstream responses. In the initial decoding of calcium signals, Ca²⁺ sensor proteins that bind Ca²⁺ and activate downstream signaling components are implicated, thereby regulating specific physiological and biochemical processes. After calcineurin B-like proteins (CBLs) sense these Ca²⁺ signatures, these proteins interact selectively with CBL-interacting protein kinases (CIPKs), thereby forming CBL/CIPK complexes, which are involved in decoding calcium signals. Therefore, specificity, diversity, and complexity are the main characteristics of the CBL-CIPK signaling system. However, additional CBLs, CIPKs, and CBL/CIPK complexes remain to be identified in plants, and the specific functions of their abiotic and biotic stress signaling will need to be further dissected. Therefore, a much-needed synthesis of recent findings is important to further the study of CBL-CIPK signaling systems. Here, we review the structure of CBLs and CIPKs, discuss the current knowledge of CBL–CIPK pathways that decode calcium signals in Arabidopsis, and link plant responses to a variety of environmental stresses with specific CBL/CIPK complexes. This will provide a foundation for future research on genetically engineered resistant plants with enhanced tolerance to various environmental stresses.     

Multifaceted Role of Salicylic Acid in Combating Cold Stress in Plants: A Review

Plants face different types of stresses, including biotic and abiotic stresses. Among various abiotic stress, low-temperature stress alters various morphological, cytological, physiological, and other biochemical processes in plants. To thrive in such condition’s plants must adopt some strategy. Out of various strategies, the approach of using plant growth regulators (PGRs) gained a prominent role in the alleviation of multiple stresses. Salicylic acid, application triggers tolerance to both biotic and abiotic stresses via regulation of various morpho-physiological, cytological, and biochemical attributes. SA is shown to alleviate and regulate the various cold-induced changes. Both endogenous and exogenously applied SA show an imperative role in the alleviation of cold-induced changes by activating multiple signaling pathways like ABA-dependent or independent pathway, Ca²⁺ signaling pathway, mitogen-activated protein kinase (MAPKs) pathway, reactive oxygen species (ROS), and reactive nitrogen species (RNS) pathways. Activation of these pathways leads to the amelioration of the cold-induced changes by increasing production of antioxidants, osmolytes, HSPs and other cold-responsive proteins like LEA, dehydrins, AFPs, PR proteins, and various other proteins. This review describes the tolerance of cold stress by SA in plants through the involvement of different stress signaling pathways.

     

Stress responses mediated by the CBL calcium sensors in plants

Calcium ions (Ca²⁺) are involved as second messenger in plant responses to a broad array of environmental stimuli, including biotic and abiotic stresses. Therefore, understanding Ca²⁺-signaling mechanisms may lead to the development of transgenic crops with enhanced tolerance to adverse environmental conditions. In order to initiate the signaling cascades and give rise to relevant cellular and physiological responses, changes in the parameters of Ca²⁺ transients should be first detected by appropriate Ca²⁺ sensors in plant cells. In this regard, elucidations of plant Ca²⁺ sensors and their target molecules are critical steps for unraveling the Ca²⁺ signal transduction pathways. Recent studies have revealed that plants possess many Ca²⁺-binding proteins with different properties, which can serve as distinct Ca²⁺ sensors. This present review mainly focuses on a family of calcineurin B-like Ca²⁺ sensors which has been most recently identified from higher plants including Arabidopsis, rice, maize and pea.     

Calmodulin-binding proteins in brain

It is now widely accepted that actions of intracellular Ca²⁺ are mediated by a four-domain Ca²⁺-binding protein, calmodulin. Brain is especially rich in calmodulin, containing about 400 mg (24 μmol) of EGTA-extractable calmodulin per kg of brain. However, only a fraction of the above amount is required for the calmodulin-activated enzymes and most of the rest may be assigned to calmodulin-binding proteins, proteins which are apparently devoid of enzyme activities but undergo Ca²⁺-dependent associations with calmodulin. Several of such proteins have been recently discovered in brain. These include a heat-labile 80 K phosphodiesterase inhibitor protein (calcineurin), a heat-stable 70 K phosphodiesterase inhibitor protein, a 50 K protein, myelin basic protein, tubulin, microtubule τ (tau) factor, a spectrin-like doublet protein (240 plus 235 K) (calspectin; fodrin) and a particle-associated 155 K protein.Functions of these calmodulin-binding proteins have not been fully elucidated yet. Some proteins may be calmodulin-regulated enzymes catalyzing yet unknown biochemical reactions, e.g. a protein phosphatase activity was found for calcineurin. Some proteins may interact with contractile elements or cytoskeleton of the cell, e.g. τ factor and calspectin interacted with tubulin and F-actin, respectively and tubulin itself is a calmodulin-binding protein. So, interesting possibilities are the regulation of the functions of cytoskeleton by calmodulin through these calmodulin-binding proteins. Regulation of microtubule assembly by Ca²⁺-dependent binding of calmodulin to tubulin and/or τ factor and possible involvement of calspectin in the mechanism regulating axonal transport of neuronal proteins have been suggested. Thus, the exploration of the regulating functions of Ca²⁺/calmodulin in brain depends largely upon the further study of the properties of these calmodulin-binding proteins.     

Calcium Signaling Network in Plants: An Overview

Calcium ion (Ca²⁺) is one of the very important ubiquitous intracellular second messenger molecules involved in many signal transduction pathways in plants. The cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt) have been found to increased in response to many physiological stimuli such as light, touch, pathogenic elicitor, plant hormones and abiotic stresses including high salinity, cold and drought. This Ca²⁺ spikes normally result from two opposing reactions, Ca²⁺ influx through channels or Ca²⁺ efflux through pumps. The removal of Ca²⁺ from the cytosol against its electrochemical gradient to either the apoplast or to intracellular organelles requires energized ‘active’ transport. Ca²⁺-ATPases and H⁺/Ca²⁺ antiporters are the key proteins catalyzing this movement. The increased level of Ca²⁺ is recognised by some Ca²⁺-sensors or calcium-binding proteins, which can activate many calcium dependent protein kinases. These kinases regulate the function of many genes including stress responsive genes, resulted in the phenotypic response of stress tolerance. Calcium signaling is also involved in the regulation of cell cycle progression in response to abiotic stress. The regulation of gene expression by cellular calcium is also crucial for plant defense against various stresses. However, the number of genes known to respond to specific transient calcium signals is limited. This review article describes several aspects of calcium signaling such as Ca²⁺ requiremant and its role in plants, Ca²⁺ transporters, Ca²⁺-ATPases, H⁺/ Ca²⁺-antiporter, Ca²⁺-signature, Ca²⁺-memory and various Ca²⁺-binding proteins (with and without EF hand).Key Words: Calcium binding proteins, Ca²⁺ channel, Ca²⁺-dependent protein kinases, Ca²⁺/H⁺ antiport, calcium memory, calcium sensors, calcium signatures, Ca²⁺-transporters, EF hand motifs, plant signal transduction