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Proton pumps produce electrical potential differences and differences in pH across the plasma membrane of cells which drive secondary ion transport through sym- and antiporters. We used the patch-clamp technique to characterize an H+-pump in the xylem parenchyma of barley roots. This cell type is of special interest with respect to xylem loading. Since it has been an ongoing debate whether xylem loading is a passive or an active process, the functional characterization of the H+-pump is of major interest in the context of previous work on ion channels through which passive salt efflux into the xylem vessels could occur. Cell-type specific features like its Ca2+ dependence were determined, that are important to interpret its physiological role and eventually to model xylem loading. We conclude that the electrogenic pump in the xylem parenchyma does not participate directly in the transfer of KCl and KNO3 to the xylem but, in combination with short-circuiting conductances, plays a crucial role in controlling xylem unloading and loading through modulation of the voltage difference across the plasma membrane. Here, our recent results on the H+ pump are put in a larger context and open questions are highlighted.Key Words: plant nutrition, H+-ATPase, anion conductance, K+ channel, electrophysiology, signaling networkThe root xylem parenchyma is of major interest with respect to nutrient (and signal) traffic between root and shoot. One of its main functions appears to be xylem loading. However, the cell walls of the vascular tissue provide apoplastic paths between xylem and phloem that represent the upward and downward traffic lanes, allowing nutrient circulation1 (Fig. 1). Therefore mechanisms for ion uptake and for ion release must exist side by side. In the last 15 years major progress has been made in the investigation of transport properties of xylem-parenchyma cells, and both uptake and release channels and transporters were identified. Today, we have good knowledge on the role of K+ and anion conductances in xylem loading with salts.2 Note, that from the functionally well characterized conductances only the molecular structure of K+ channels is known. In contrast, many transporters are identified on the molecular level, but functional data are scarce.Open in a separate windowFigure 1Distribution of tissues in the periphery of the stele. The stippled area marks the region from which early metaxylem protoplasts originated. E, Endodermis with Casparian strip; eMX, ‘early’ metaxylem vessel; IMX, ‘late’ metaxylem vessel; Mph, metaphloem (sieve tube); Pph, protophloem (sieve tube); P, pericycle; Cx, cortex. Symplasmic and apoplasmic transport routes are indicated in red and black, respectively. The Casparian strip prevents apoplastic transport into the stele. Plasmodesmata are shown exemplarily for the indicated symplastic pathway. All cells of the symplast are connected via plasmodesmata. Sites of active uptake into the root symplast and of release into the stelar apoplast are indicated by a black and an orange arrow. Modified from Wegner and Raschke, 1994.3A challenging question to deal with was the dispute about xylem loading with ions being a passive or active process. While it is clear that energy through electrogenic H+ efflux is needed to take up nutrient ions from the soil against their electrochemical gradient into the cortical symplast, it has been a matter of debate if ion release into xylem vessels also is energy-linked or if the electrochemical potentials of ions are raised high enough to allow a thermodynamically passive flux.2,3 The Casparian strip prohibits apoplastic transport of nutrients into the stele and electrically insulates the stelar from the cortical apoplast. Therefore the electrical potential difference of the cells in the xylem parenchyma could be independent from the cortical potential difference but be subject to control, for instance, from the shoot.4 Indeed, evidence points to xylem loading as a second control point in nutrient transfer to the shoot.5,6 The identification and characterization of K+ and anion conductances clearly showed that release of KCl and KNO3 into the xylem can be passive through voltage-dependent ion channels.2,3,79 No need appeared for a pump energizing the transfer of salts to the xylem.However, H+ pumps are ubiquitous. H+-ATPases are encoded by a multigene family and heterologous expression in yeast showed that isoforms have distinct enzymatic properties.10,11 As the example of the amino acid transporter AAP6 from the xylem parenchyma shows, a cell-type specific functional characterization of transporters is essential to draw conclusions on their physiological role. AAP6 is the only member of a multigene family with an affinity for aspartate in the physiologically relevant range. The actual apoplastic concentration of amino acids and the pH will determine what is transported in vivo.12,13 Xylem-parenchyma cells of barley roots were strongly labelled by antibodies against the plasma membrane H+-ATPase.14 In a recent publication in Physiologia Plantarum we report the functional analysis of the electrogenic pump from the plasma membrane of xylem parenchyma from barley roots that was done with the patch-clamp technique after specific isolation of protoplasts from this cell type. It displayed characteristics of an H+-ATPase: current-voltage relationships were characteristic for a ‘rheogenic’ pump15 and currents were stimulated by fusicoccin or by an enlarged transmembrane pH gradient and inhibited by dicyclohexylcarbodiimide (DCCD). Importantly, it also showed distinct characteristics. Neither intracellular pH nor the intracellular Ca2+ concentration affected its activity. Noteworthy, K+ and anion conductances from the same cell type are controlled by intracellular [Ca2+]7,9 (Fig. 2). It was proposed that the effect of abscisic acid (ABA) on anion conductances is mediated via an increase in the cytosolic Ca2+ concentration.16 Very likely stelar H+ pumps are stimulated by ABA.17 Thus, a Ca2+ independent control has to be hypothesized in this case.Open in a separate windowFigure 2Control of ion conductances in the plasma membrane of xylem-parenchyma cells. Arrowheads indicate stimulation and bars indicate inhibition by an increase in cytosolic [Ca2+],7,9,16 by ABA,16,17,21 by cytosolic and apoplastic acidification,4,22 by G-proteins23 and by an increase in apoplastic [K+]7 and [NO3].24 Apoplastic [K+] and [NO3] modify the voltage dependence exerting negative feedback on K+ efflux and a positive feedback on NO3 efflux. Abscisic acid has an immediate effect on ion channel activity, most likely via [Ca2+], and causes a change in gene expression as indicated by circles (up) and bars (down). ABA perception is not clear. A Ca2+ influx could occur through a hyperpolarization activated cation conductance (HACC).16,25 Cation transporters are NORC, nonselective cation conductance, KORC, K+-selective outwardly rectifying conductance (=SKOR8), and KIRC, K+-selective inwardly rectifying conductance, and anion conductances with different voltage-dependencies and gating characteristics are X-QUAC, quickly activating anion conductance, X-SLAC, slowly activating anion conductance, and X-IRAC, inwardly rectifying anion channel.2,3,9,16,26 Transported ions and direction of flux are plotted.To date, we know that besides Ca2+ and abscisic acid also the pH, nonhydrolyzable GTP analogs and extracellular NO3 and K+ affect membrane transport capacities of root xylem-parenchyma cells (Fig. 2). Other control mechanisms by metabolites, the redox potential and phytohormones have to be included, especially if they represent signals in xylem loading or root-shoot communication. The composition of the xylem sap changes during the course of a day, depending on nutrient supply and various stresses, and the apoplastic ion concentration is considered to be an important factor in ion circulation.6,18,19 ABA is such a signal. It is known to increase solute accumulation within the root by inhibiting release of ions into the xylem.17 Any change in transport activity has an impact on the membrane potential. This again determines whether salt release or uptake takes place. Passive salt release is restricted to a limited range of membrane potentials in which conductances for anions and cations are active simultaneously, that is with depolarization. Negative membrane voltages will be required for reabsorption of NO3 by a putative NO3/H+-symporter and for the uptake of K+ and amino acids.3,13 As shown in our recent paper, the balance between the activities of the H+-pump and the anion conductances could affect the position between a depolarized and a hyperpolarized state of the parenchymal membrane. Thus, H+ pump activity is crucial in membrane voltage control. Furthermore, the simultaneous activities of H+ pumps and anion conductances make the generation of a high pH gradient possible, whilst maintaining electroneutrality. The proton gradient could be used for ion transport through cotransporters and antiporters as suggested for the loading of borate into the xylem through the boron transporter BOR1.20 So we are on the way to decipher xylem loading in roots and this exciting field will also provide information about small-scale nutrient cycling and root-shoot communication. To determine how the activities of pumps, channels and transporters are adjusted among each other is the next challenge. Further insight has to be obtained by experimentation as well as by biophysical modeling.  相似文献   

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一步法快速质粒鉴定方法   总被引:1,自引:0,他引:1  
对《分子克隆实验指南》(第三版)中的牙签法小量制备质粒DNA的方法做了改进,减少了 加样次数,免去冰浴、离心等步骤,改进后的方法在琼脂糖凝胶电泳前仅需一步加样和加热过程, 提高了实验效率。同时结合使用多孔道移液器和96孔板,更适合于在高通量筛选中鉴定阳性克 隆。实验对改进后的方法与《分子克隆实验指南》上的方法进行了比较,表明“一步法”的检测效 果、准确率与重复性均有到较好的结果。  相似文献   

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A mathematical model simulation was performed to estimate the amount of radioactivity in plasma, normal tissues, and tumor tissue through three delivery approaches: one step radiolabeled monoclonal antibody (MAb) CC49 i.v. bolus injection, two step method with biotin conjugated CC49 i.v. bolus injection followed 72 hours later by i.v. bolus radiolabeled streptavidin injection, and gene therapy method to express biotin on the tumor cell surface followed by i.v. bolus radiolabeled streptavidin injection. The mathematical model was built based on a system of ordinary differential equations consisting of inputs and outputs of model components in plasma, normal tissues, and tumor tissue. Through computer modeling, we calculated concentrations of each component for plasma, tumor and normal tissues at various time points. Radioactivity ratios of tumor to plasma and tumor to normal tissues increased with time. The increase of tumor to normal tissue ratios was much faster for the gene therapy approach than for single step and two step approaches, e.g., a ratio of 24.26 vs. 2.06 and 6.24 at 72 hours after radioligand injection. Radioactivity ratios predicted by the model varied with the amount of radioactivity injected and the time interval between injections. The model could be used to evaluate different radioimmunotherapy strategies and to predict radioactivity biodistribution using other receptor-ligand systems.  相似文献   

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Assumptions about the costs of character change, coded in the form of a step matrix, determine most-parsimonious inferences of character evolution on phylogenies. We present a graphical approach to exploring the relationship between cost assumptions and evolutionary inferences from character data. The number of gains and losses of a binary trait on a phylogeny can be plotted over a range of cost assumptions, to reveal the inflection point at which there is a switch from more gains to more losses and the point at which all changes are inferred to be in one direction or the other. Phylogenetic structure in the data, the tree shape, and the relative frequency of states among the taxa influence the shape of such graphs and complicate the interpretation of possible permutation-based tests for directionality of change. The costs at which the most-parsimonious state of each internal node switches from one state to another can also be quantified by iterative ancestral-state reconstruction over a range of costs. This procedure helps identify the most robust inferences of change in each direction, which should be of use in designing comparative studies.  相似文献   

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一步法制备羧甲基茯苓多糖的工艺研究   总被引:4,自引:0,他引:4  
本实验对在有机溶剂中一步法半合成羧甲基茯苓多糖的合成条件进行了研究。结果表明,乙醇是作为羧甲基化反应的合适介质。反应温度提高能加快反应速度;反应时间延长能提高取代度。茯苓多糖葡萄糖当量与氢氧化钠和一氯乙酸的摩尔比调配适当,能减少副产物羟乙酸钠的产生。  相似文献   

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