Effects of ABA and CaCl2 on GABA accumulation in fava bean germinating under hypoxia-NaCl stress

Effects of exogenous abscisic acid (ABA) and CaCl₂ on γ-aminobutyric acid (GABA) accumulation of germinated fava bean under hypoxia-NaCl stress were investigated. Exogenous ABA resulted in the enhancement of glutamate decarboxylase (GAD) and diamine oxidase (DAO) activity as well as GABA content in cotyledon and shoot. CaCl₂ increased both enzyme activities in shoot and GABA content in cotyledon and shoot. ABA downregulated GAD expression in cotyledon and radicle, while upregulated that in shoot; it also upregulated DAO expression in each organ. CaCl₂ upregulated GAD expression in cotyledon, while downregulated that in radicle. However, it upregulated DAO expression in shoot, downregulated that in radicle. ABA inhibitor fluridon and ethylenediaminetetraacetic acid inhibited GAD and DAO activities significantly so that inhibited GABA accumulation through reducing ABA biosynthesis and chelating Ca²⁺, respectively. However, they upregulated GAD and DAO expression in varying degrees. These results indicate that ABA and Ca²⁺ participate in GABA biosynthesis in fava bean during germination under hypoxia-NaCl stress.     

Cell biological mechanism for triggering of ABA accumulation under water stress inVicia faba leaves

Water stress-induced ABA accumulation is a cellular signaling process from water stress perception to activation of genes encoding key enzymes of ABA biosynthesis, of which the water stress-signal perception by cells or triggering mechanism of the ABA accumulation is the center in the whole process of ABA related-stress signaling in plants. The cell biological mechanism for triggering of ABA accumulation under water stress was studied in leaves ofVicia faba. Mannitol at 890 mmol ·kg^-1 osmotic concentration induced an increase of more than 5 times in ABA concentration in detached leaf tissues, but the same concentration of mannitol only induced an increase of less than 40 % in ABA concentration in protoplasts. Like in detached leaf tissues, ABA concentration in isolated cells increased more than 10 times under the treatment of mannitol at 890 mmol · kg^-1 concentration, suggesting that the interaction between plasmalemma and cell wall was essential to triggering of the water stress-induced ABA accumulation. Neither Ca²⁺-chelating agent EGTA nor Ca²⁺channel activator A23187 nor the two cytoskeleton inhibitors, colchicine and cytochalasin B, had any effect on water stress-induced ABA accumulation. Interestingly water stress-induced ABA accumulation was effectively inhibited by a non-plasmalemma-permeable sulfhydryl-modifier PCMBS (p-chloromercuriphenyl-sulfonic acid), suggesting that plasmalemma protein(s) may be involved in the triggering of water stress-induced ABA accumulation, and the protein may contain sulfhydryl group at its function domain.     

Calcium and copper transport ATPases: analogies and diversities in transduction and signaling mechanisms

The calcium transport ATPase and the copper transport ATPase are members of the P-ATPase family and retain an analogous catalytic mechanism for ATP utilization, including intermediate phosphoryl transfer to a conserved aspartyl residue, vectorial displacement of bound cation, and final hydrolytic cleavage of Pi. Both ATPases undergo protein conformational changes concomitant with catalytic events. Yet, the two ATPases are prototypes of different features with regard to transduction and signaling mechanisms. The calcium ATPase resides stably on membranes delimiting cellular compartments, acquires free Ca²⁺ with high affinity on one side of the membrane, and releases the bound Ca²⁺ on the other side of the membrane to yield a high free Ca²⁺ gradient. These features are a basic requirement for cellular Ca²⁺ signaling mechanisms. On the other hand, the copper ATPase acquires copper through exchange with donor proteins, and undergoes intracellular trafficking to deliver copper to acceptor proteins. In addition to the cation transport site and the conserved aspartate undergoing catalytic phosphorylation, the copper ATPase has copper binding regulatory sites on a unique N-terminal protein extension, and has also serine residues undergoing kinase assisted phosphorylation. These additional features are involved in the mechanism of copper ATPase intracellular trafficking which is required to deliver copper to plasma membranes for extrusion, and to the trans-Golgi network for incorporation into metalloproteins. Isoform specific glyocosylation contributes to stabilization of ATP7A copper ATPase in plasma membranes.     

Ca~(2+)信号在植物与环境微生物互相作用中的分子调控机制

自然界植物与环境微生物之间的相互关系除了胁迫以外,同时也有互利互惠的共生互作关系。无论是能对植物造成胁迫伤害的植物-病原菌互作体系,还是能够为植物提供营养的植物-微生物共生互作体系,其细胞信号转导通路中Ca~(2+)信号的分子调控对两种互作体系都有着非常重要的作用。该文对近年来国内外有关植物-病原菌和植物-微生物互作体系在细胞信号转导过程中Ca~(2+)信号上游的分子调控机制分别进行了综述。     

Ca signaling and STIM1

An increase in the intracellular calcium ion concentration ([Ca²⁺]) impacts a diverse range of cell functions, including adhesion, motility, gene expression and proliferation. Elevation of intracellular calcium ion (Ca²⁺) regulates various cellular events after the stimulation of cells. Initial increase in Ca²⁺ comes from the endoplasmic reticulum (ER), intracellular storage space. However, the continuous influx of extracellular Ca²⁺ is required to maintain the increased level of Ca²⁺ inside cells. Store-operated Ca²⁺ entry (SOCE) manages this process, and STIM1, a newly discovered molecule, has a unique and essential role in SOCE. STIM1 can sense the exhaustion of Ca²⁺ in the ER, and activate the SOC channel in the plasma membrane, leading to the continuous influx of extracellular Ca²⁺. STIM1 senses the status of the intracellular Ca²⁺ stores via a luminal N-terminal Ca²⁺-binding EF-hand domain. Dissociation of Ca²⁺ from this domain induces the clustering of STIM1 to regions of the ER that lie close to the plasma membrane, where it regulates the activity of the store-operated Ca²⁺ channels/entry (calcium-release-activated calcium channels/entry). In this review, we summarize the mechanism by which STIM1 regulates SOCE, and also its role in the control of mast cell functions and allergic responses.     

Regulation of CRAC channels by protein interactions and post-translational modification

Store-operated Ca²⁺ entry (SOCE) is a widespread mechanism to elevate the intracellular Ca²⁺ concentrations and stimulate downstream signaling pathways affecting proliferation, secretion, differentiation and death in different cell types. In immune cells, immune receptor stimulation induces intracellular Ca²⁺ store depletion that subsequently activates Ca²⁺-release-activated-Ca²⁺ (CRAC) channels, a prototype of store-operated Ca²⁺ (SOC) channels. CRAC channel opening leads to activation of diverse downstream signaling pathways affecting proliferation, differentiation, cytokine production and cell death. Recent identification of STIM1 as the endoplasmic reticulum Ca²⁺ sensor and Orai1 as the pore subunit of CRAC channels has provided the much-needed molecular tools to dissect the mechanism of activation and regulation of CRAC channels. In this review, we discuss the recent advances in understanding the associating partners and posttranslational modifications of Orai1 and STIM1 proteins that regulate diverse aspects of CRAC channel function.     

Ca-induced fusion of cardiolipin/phosphatidylcholine vesicles monitored by mixing of aqueous contents

The kinetics of Ca²⁺-induced fusion of large (0.1 μm) unilamellar cardiolipin/phosphatidylcholine (1:1) vesicles have been investigated by continuous monitoring of the mixing of the aqueous vesicle contents. In parallel, release of vesicle contents to the external medium has been followed. Initial fusion of the vesicles is non-leaky, release of vesicle contents being largely a secondary phenomenon. The minimal Ca²⁺ concentration required for fusion in this system is approx. 9 mM. At higher Ca²⁺ concentrations fusion is extremely fast, occurring on the time scale of seconds.     

Ca(2+) signaling mechanisms of cell survival and cell death: an introduction

Ca²⁺ regulates many steps in cell death mechanisms, and is potentially involved in all types of cell death. Moreover, virtually all elements of the cellular Ca²⁺ toolbox seem to contribute to remodeling of the Ca²⁺ signaling machinery during cell death processes. As expected from the ubiquitous nature of Ca²⁺ signaling, these mechanisms are operative in all cell types, and their malfunction may lead to a wide diversity of pathological implications. The contributions in this Special Issue deal with many different aspects of the relation between Ca²⁺ signaling and cell death. They illustrate the complexity of this relation, and importantly they give an outlook on potential new therapeutic targets for treatment of diseases connected to defects in cell death pathways.     

Interdomain interactions within ryanodine receptors regulate Ca2+ spark frequency in skeletal muscle.

DP4 is a 36-residue synthetic peptide that corresponds to the Leu(2442)-Pro(2477) region of RyR1 that contains the reported malignant hyperthermia (MH) mutation site. It has been proposed that DP4 disrupts the normal interdomain interactions that stabilize the closed state of the Ca(2)+ release channel (Yamamoto, T., R. El-Hayek, and N. Ikemoto. 2000. J. Biol. Chem. 275:11618-11625). We have investigated the effects of DP4 on local SR Ca(2)+ release events (Ca(2)+ sparks) in saponin-permeabilized frog skeletal muscle fibers using laser scanning confocal microscopy (line-scan mode, 2 ms/line), as well as the effects of DP4 on frog SR vesicles and frog single RyR Ca(2)+ release channels reconstituted in planar lipid bilayers. DP4 caused a significant increase in Ca(2)+ spark frequency in muscle fibers. However, the mean values of the amplitude, rise time, spatial half width, and temporal half duration of the Ca(2)+ sparks, as well as the distribution of these parameters, remained essentially unchanged in the presence of DP4. Thus, DP4 increased the opening rate, but not the open time of the RyR Ca(2)+ release channel(s) generating the sparks. DP4 also increased [(3)H]ryanodine binding to SR vesicles isolated from frog and mammalian skeletal muscle, and increased the open probability of frog RyR Ca(2)+ release channels reconstituted in bilayers, without changing the amplitude of the current through those channels. However, unlike in Ca(2)+ spark experiments, DP4 produced a pronounced increase in the open time of channels in bilayers. The same peptide with an Arg(17) to Cys(17) replacement (DP4mut), which corresponds to the Arg(2458)-to-Cys(2458) mutation in MH, did not produce a significant effect on RyR activation in muscle fibers, bilayers, or SR vesicles. Mg(2)+ dependence experiments conducted with permeabilized muscle fibers indicate that DP4 preferentially binds to partially Mg(2)+-free RyR(s), thus promoting channel opening and production of Ca(2)+ sparks.     

Role of platelet plasma membrane Ca-ATPase in health and disease

Platelets have essential roles in both health and disease. Normal platelet function is required for hemostasis. Inhibition of platelet function in disease or by pharmacological treatment results in bleeding disorders. On the other hand, hyperactive platelets lead to heart attack and stroke. Calcium is a major second messenger in platelet activation, and elevated intracellular calcium leads to hyperactive platelets. Elevated platelet calcium has been documented in hypertension and diabetes; both conditions increase the likelihood of heart attack and stroke. Thus, proper regulation of calcium metabolism in the platelet is extremely important. Plasma membrane Ca(2+)-ATPase (PMCA) is a major player in platelet calcium metabolism since it provides the only significant route for calcium efflux. In keeping with the important role of calcium in platelet function, PMCA is a highly regulated transporter. In human platelets, PMCA is activated by Ca(2+)/calmodulin, by cAMP-dependent phosphorylation and by calpain-dependent removal of the inhibitory peptide. It is inhibited by tyrosine phosphorylation and calpain-dependent proteolysis. In addition, the cellular location of PMCA is regulated by a PDZ-domain-dependent interaction with the cytoskeleton during platelet activation. Rapid regulation by phosphorylation results in changes in the rate of platelet activation, whereas calpain-dependent proteolysis and interaction with the cytoskeleton appears to regulate later events such as clot retraction. In hypertension and diabetes, PMCA expression is upregulated while activity is decreased, presumably due to tyrosine phosphorylation. Clearly, a more complete understanding of PMCA function in human platelets could result in the identification of new ways to control platelet function in disease states.     

CPK12: A Ca2+-dependent protein kinase balancer in abscisic acid signaling

Ca²⁺ is believed to be a critical second messenger in ABA signal transduction. Ca²⁺-dependent protein kinases (CDPKs) are the best characterized Ca²⁺ sensors in plants. Recently, we identified an Arabidopsis CDPK member CPK12 as a negative regulator of ABA signaling in seed germination and post-germination growth, which reveals that different members of the CDPK family may constitute a regulation loop by functioning positively and negatively in ABA signal transduction. We observed that both RNA interference and overexpression of CPK12 gene resulted in ABA-hypersensitive phenotypes in seed germination and post-germination growth, suggesting a high complexity of the CPK12-mediated ABA signaling pathway. CPK12 stimulates a negative ABA-signaling regulator (ABI2) and phosphorylates two positive ABA-signaling regulators (ABF1 and ABF4), which may partly explain the ABA hypersensitivity induced by both downregulation and upregulation of CPK12 expression. Our data indicate that CPK12 appears to function as a balancer in ABA signal transduction in Arabidopsis.     

Ins(1,4,5)P3 receptor-mediated Ca2+ signaling and autophagy induction are interrelated

The role of intracellular Ca2+ signaling in starvation-induced autophagy remains unclear. Here, we examined Ca2+ dynamics during starvation-induced autophagy and the underlying molecular mechanisms. Tightly correlating with autophagy stimulation, we observed a remodeling of the Ca2+ signalosome. First, short periods of starvation (1 to 3 h) caused a prominent increase of the ER Ca2+-store content and enhanced agonist-induced Ca2+ release. The mechanism involved the upregulation of intralumenal ER Ca2+-binding proteins, calreticulin and Grp78/BiP, which increased the ER Ca2+-buffering capacity and reduced the ER Ca2+ leak. Second, starvation led to Ins(1,4,5)P3R sensitization. Immunoprecipitation experiments showed that during starvation Beclin 1, released from Bcl-2, first bound with increasing efficiency to Ins(1,4,5)P3Rs; after reaching a maximal binding after 3 h, binding, however, decreased again. The interaction site of Beclin 1 was determined to be present in the N-terminal Ins(1,4,5)P3-binding domain of the Ins(1,4,5)P3R. The starvation-induced Ins(1,4,5)P3R sensitization was abolished in cells treated with BECN1 siRNA, but not with ATG5 siRNA, pointing toward an essential role of Beclin 1 in this process. Moreover, recombinant Beclin 1 sensitized Ins(1,4,5)P3Rs in 45Ca2+-flux assays, indicating a direct regulation of Ins(1,4,5)P3R activity by Beclin 1. Finally, we found that Ins(1,4,5)P3R-mediated Ca2+ signaling was critical for starvation-induced autophagy stimulation, since the Ca2+ chelator BAPTA-AM as well as the Ins(1,4,5)P3R inhibitor xestospongin B abolished the increase in LC3 lipidation and GFP-LC3-puncta formation. Hence, our results indicate a tight and essential interrelation between intracellular Ca2+ signaling and autophagy stimulation as a proximal event in response to starvation.     

Activation of the human red cell calcium ATPase by calcium pretreatment

Some kinetic parameters of the human red cell Ca²⁺-ATPase were studied on calmodulin-free membrane fragments following preincubation at 37°C. After 30 min treatment with EGTA(1 mm) plus dithioerythritol (1 mm), a V _max of about 0.4 μmol P_i/mg × hr and a K _s of 0.3 μm Ca²⁺ were found. When Mg²⁺ (10 mm) or Ca²⁺(10 μm) were also added during preincubation, V _maxbut not Kwas altered. Ca²⁺ was more effective than Mg²⁺, thus increasing V _max to about 1.3 μmol P_i/mg × hr. The presence of both Ca²⁺ and Mg²⁺ during pretreatment decreasedKto 0.15 μm, while having no apparent effect on V _max. Conversely, addition of ATP (2 mm) with either Ca²⁺ or Ca²⁺ plus Mg²⁺increased V_max without affecting K. Preincubation with Ca²⁺ for periods longer than 30 min further increased V_maxand reduced Kto levels as low as found with calmodulin treatment. The Ca²⁺ activation was not prevented by adding proteinase inhibitors (iodoacetamide, 10 mm; leupeptin, 200 μm; pepstatinA, 100 μm; phenylmethanesulfonyl fluoride, 100 μm). The electrophoretic pattern of membranes preincubated with or without Mg²⁺, Ca²⁺ or Ca²⁺ plus Mg²⁺ did not differ significantly from each other. Moreover, immunodetection of Ca²⁺-ATPase by means of polyclonal antibodiesrevealed no mobility change after the various treatments. The above stimulation was not altered by neomycin (200 μm), washing with EGTA (5 mm) or by both incubating and washing with delipidized serum albumin (1 mg/ml), or omitting dithioerythritol from the preincubation medium. On the other hand, the activation elicited by Ca²⁺ plus ATP in the presence of Mg²⁺ was reduced 25–30% by acridine orange (100 μm), compound 48/80 (100 μm) or leupeptin (200 μm) but not by dithio-bis-nitrobenzoic acid (1 mm). The fluorescence depolarization of 1,6-diphenyl-and l-(4-trimethylammonium phenyl)-6-phenyl 1,3,5-hexatriene incorporated into membrane fragments was not affected after preincubating under the different conditions. The results show that proteolysis, fatty acid production, an increased phospholipid metabolism or alteration of membrane fluidity are not involved in the Ca²⁺ effect. Ca²⁺ preincubation may stimulate the Ca²⁺-ATPase activity by stabilizing or promoting the E₁ conformation.     

干旱胁迫下植物根源化学信号研究进展

土壤干旱胁迫诱导植物根系产生根源化学信号,经运输系统长距离传输到地上部分,降低气孔导度,抑制蒸腾作用,从而提高植物的水分利用效率。根源化学信号包括脱落酸(ABA)、细胞分裂素(CTK)、生长素、木质部pH值和钙离子(Ca²⁺)等,其中以ABA为主的植物根源信号通路研究得最为广泛和深入。总结了几种主要的化学根源信号物质的基本性质、主要功能和调节机制,重点对这些信号参与气孔行为、差别基因表达和生长发育方面的研究进展进行了综述。由于干旱条件下植物根源信号反应涉及到从分子到群体的一系列复杂过程,各种信号的生理效应呈现交互作用、耦合发生的特点,今后的热点领域将集中在研究交互网络中合成的的关键物质和揭示这些物质在分子及生理水平上的作用机理上。根源化学信号研究正朝向"以分子和生理研究为基础、不同尺度的结构和功能耦合"的方向发展。     

Anion channels as central mechanisms for signal transduction in guard cells and putative functions in roots for plant-soil interactions

In higher plants anion channels have recently been suggested to play key roles in controlling cellular functions, including turgor- and osmoregulation, stomatal movements, anion transport, signal transduction and possibly also signal propagation. In guard cells and roots, physiological functions of anion channels have been proposed which will be discussed here. In initial investigations it was proposed that anion channels in the plasma membrane of guard cells provide a prominent control mechanism for stomatal closing. The proposed model suggests that anion channel activation and the resulting anion efflux from guard cells cause membrane depolarization, thereby driving K⁺ efflux through outward-rectifying K⁺ channels required for stomatal closing. This article provides a brief review of new and recent insights into the molecular properties and cell biological functions of anion channels in guard cells. Furthermore, recently implicated putative functions of anion channels in roots during salt stress, xylem loading and Al³⁺ tolerance are addressed.     

Phospholipase C is required for the control of stomatal aperture by ABA

The calcium-releasing second messenger inositol 1,4,5-trisphosphate is involved in the regulation of stomatal aperture by ABA. In other signalling pathways, inositol 1,4,5-trisphosphate is generated by the action of phospholipase C. We have studied the importance of phospholipase C in guard cell ABA-signalling pathways. Immunolocalisation of a calcium-activated phospholipase C confirmed the presence of phospholipase C in tobacco guard cells. Transgenic tobacco plants with considerably reduced levels of phospholipase C in their guard cells were only partially able to regulate their stomatal apertures in response to ABA. These results suggest that phospholipase C is involved in the amplification of the calcium signal responsible for reductions in stomatal aperture in response to ABA. As full ABA-induced inhibition of stomatal opening was not observed, our results support a role for the action of other calcium-releasing second messengers in the guard cell ABA-signalling pathway. It is not known whether these different calcium-releasing second messengers act in the same or parallel ABA-signalling pathways.