Localization of Hydrogen Ion and Chloride Ion Fluxes in Nitella

Alternating bands of acid and base formation have been detected along the length of the internodal cell of Nitella clavata when it is illuminated, while in the dark this phenomenon is minimal. Chloride influx occurs only or largely in the acid-extruding regions, and this is also a light-dependent ion movement. Chloride efflux is slightly dependent on illumination and is not localized as are H⁺ efflux and Cl^- influx. The results obtained support Kitasato's (1968) proposal that a large passive H⁺ influx is balanced by an active efflux of this ion. Transport mechanisms suggested by the correlations of Cl^- and HCO₃^- influxes with H⁺ extrusion are discussed.     

Metabolic Fluxes in an Illuminated Arabidopsis Rosette

Photosynthesis is the basis for life, and its optimization is a key biotechnological aim given the problems of population explosion and environmental deterioration. We describe a method to resolve intracellular fluxes in intact Arabidopsis thaliana rosettes based on time-dependent labeling patterns in the metabolome. Plants photosynthesizing under limiting irradiance and ambient CO₂ in a custom-built chamber were transferred into a ¹³CO₂-enriched environment. The isotope labeling patterns of 40 metabolites were obtained using liquid or gas chromatography coupled to mass spectrometry. Labeling kinetics revealed striking differences between metabolites. At a qualitative level, they matched expectations in terms of pathway topology and stoichiometry, but some unexpected features point to the complexity of subcellular and cellular compartmentation. To achieve quantitative insights, the data set was used for estimating fluxes in the framework of kinetic flux profiling. We benchmarked flux estimates to four classically determined flux signatures of photosynthesis and assessed the robustness of the estimates with respect to different features of the underlying metabolic model and the time-resolved data set.     

Strategies for Manipulating Metabolic Fluxes in Biotechnology

Strategies for biotechnologically manipulating metabolic fluxes are critically examined in relation to a model system. The common idea of first identifying the rate-limiting enzyme in the biosynthetic pathway to a desired end-product, and then increasing its activity, is shown to be completely ineffective; such manipulation typically produces only trivial changes in flux. Manipulating the activities of all of the enzymes in a biosynthetic pathway by amounts calculated to increase a desired flux while leaving all other fluxes and all concentrations unchanged is potentially effective, and can be applied to any system without regard to its regulatory design. however, it requires accurate knowledge of the initial state of the system and the ability to make precise changes to numerous activities. The classical information about the regulatory mechanisms that exist in living organisms suggests that one can make much simpler manipulations, involving only the steps that remove the desired end-product, with almost equally satisfactory results.     

Effects of salinity on turgor pressure and fertility in Tolypella (Characeae)

We present the first experimental results on salinity tolerance and regulation mechanisms in the genus Tolypella. The two species investigated, T. nidifica and T. glomerata, regulate turgor pressure with almost complete effectiveness by adjustment of K+ and CT concentrations. Sucrose is also involved. The mechanism is basically identical to the mechanism of turgor pressure regulation previously identified in representatives of the genera Chara and Lamprothamnium. Since Chara and Lamprothamnium on the one hand and Tolypella on the other belong to different phylogenetic branches that separated early in the geological history of the Characeae, the K+ regulation mechanism can be assumed to represent an ancient pattern derived from a salt-tolerant common ancestor. Furthermore, our experiments provide evidence that salinity is a limiting factor for fertility in both T. nidifica and T. glomerata. Although the onset of gametangia covers the whole range of salinities tested here (0–29 psu), 12psu was the inhibitory level for the formation of mature oospores. Fertilization is probably disturbed by an increase in salinity. An inability to reproduce sexually under euryhaline conditions could explain why the distribution of the two species is restricted to oligo- and mesohaline environments, despite the wide range of salinity tolerance of their vegetative apparatus.     

Estimating Relative Changes of Metabolic Fluxes

Fluxes are the central trait of metabolism and Kinetic Flux Profiling (KFP) is an effective method of measuring them. To generalize its applicability, we present an extension of the method that estimates the relative changes of fluxes using only relative quantitation of ¹³C-labeled metabolites. Such features are directly tailored to the more common experiment that performs only relative quantitation and compares fluxes between two conditions. We call our extension rKFP. Moreover, we examine the effects of common missing data and common modeling assumptions on (r)KFP, and provide practical suggestions. We also investigate the selection of measuring times for (r)KFP and provide a simple recipe. We then apply rKFP to ¹³C-labeled glucose time series data collected from cells under normal and glucose-deprived conditions, estimating the relative flux changes of glycolysis and its branching pathways. We identify an adaptive response in which de novo serine biosynthesis is compromised to maintain the glycolytic flux backbone. Together, these results greatly expand the capabilities of KFP and are suitable for broad biological applications.     

The Effect of Dio-9 on Photosynthesis and Ion Transport in Nitella, Tolypella, and Hydrodictyon

The effect of Dio-9, on photosynthesis and active and passiveion transport at the plasmalemma has been investigated in Nitellatranslucens, Tolypella intricata, and Hydrodictyon africanum.The active K influx and the coupled active Na efflux were moreinhibited by this inhibitor of energy transfer in phosphorylationthan was photosynthesis. The active Cl influx, the associateddownhill cation influxes, and passive ion fluxes were inhibitedby Dio-9, to a smaller extent than was photosynthesis. Cl influxin the light was often stimulated at concentrations of Dio-9,which inhibited photosynthesis. It was concluded that the activeK influx and Na efflux require ATP, while the active Cl influxdoes not. Possible links between the Cl influx and electrontransport and intermediates of photophosphorylation, and possibleinhibition by Dio-9 of the transport ATPase in the plasmalemmaare discussed.     

Control of Ion Fluxes in Stomatal Guard Cells

Opening and closing of the stomatal pore is associated with very large changes in K-salt accumulation in stomatal guard cells. This review discusses the ionic relations of guard cells in relation to the general pattern of transport processes in plant cells, in plasmalemma and tonoplast, involving primary active transport of protons, proton-linked secondary active transport, and a number of gated ion channels. The evidence available suggests that the initiation of stomatal opening is regulated through the uptake mechanisms, whereas initiation of stomatal closing is regulated by control of ion efflux at the plasmalemma, and of fluxes to and from the vacuole. In response to a closing signal there are large transient increases in efflux of both Cl^? (or Br^?) and Rb⁺ (K⁺) at the plasmalemma, with also a probable increase in anion flux from vacuole to cytoplasm and decrease in anion flux from cytoplasm to vacuole. A speculative hypothetical sequence of events is discussed, by which the primary response to a closing signal is an increase in Ca²⁺ influx at the plasmalemma, producing depolarisation and increase in cytoplasmic Ca²⁺. The consequent opening of Ca²⁺-sensitive Cl^? channels, and voltage-sensitive K⁺ channels (also Ca²⁺-sensitive?) in the plasmalemma, and of a Ca²⁺-sensitive nonspecific channel in the tonoplast, could produce the flux effects identified by the tracer work; this speculation is also consistent with the Ca²⁺-sensitivity of the response to closing signals and with evidence from patch clamping that such channels exist in at least some plant cells, though not yet all shown in guard cells.     

Determination of Ion Content and Ion Fluxes in the Halotolerant Alga Dunaliella salina

A method to determine intracellular cation contents in Dunaliella by separation on cation-exchange minicolumns is described. The separation efficiency of cells from extracellular cations is over 99.9%; the procedure causes no apparent perturbation to the cells and can be applied to measure both fluxes and internal content of any desired cation. Using this technique it is demonstrated that the intracellular averaged Na⁺, K⁺, and Ca²⁺ concentrations in Dunaliella salina cultured at 1 to 4 molar NaCl, 5 millimolar K⁺, and 0.3 millimolar Ca²⁺ are 20 to 100 millimolar, 150 to 250 millimolar, and 1 to 3 millimolar, respectively. The intracellular K⁺ concentration is maintained constant over a wide range of media K⁺ concentrations (0.5-10 millimolar), leading to a ratio of K⁺ in the cells to K⁺ in the medium of 10 to 1,000. Severe limitation of external K⁺, induces loss of K⁺ and increase in Na⁺ inside the cells. The results suggest that Dunaliella cells possess efficient mechanisms to eliminate Na⁺ and accumulate K⁺ and that intracellular Na⁺ and K⁺ concentrations are carefully regulated. The contribution of the intracellular Na⁺ and K⁺ salts to the total osmotic pressure of cells grown at 1 to 4 molar NaCl, is 5 to 20%.     

Measurements of Ion Concentrations and Fluxes in Dunaliella parva

Measurements of K⁺, Na⁺, and Cl^– were made on a halotolerantstrain of Dunaliella growing at 500 mM NaCl, 25 ?C, and a relativelylow light intensity (6000 Lx). Much effort was spent in searchingfor a means of measuring the extracellular volume of fluid trappedbetween the cells of centrifuged pellets. All of the sugarstried as markers were rejected because they were found to bedigested in the cell suspension. The most suitable marker wasfound to be [¹⁴C]polyethylene glycol² (mol. wt. 4000); althoughthis substance was apparently adsorbed to the cell exterior,it was found possible to correct for adsorption and then obtaina reasonable figure for the trapped fluid. The final concentrationsof cell K⁺ and Na⁺ were 128 ? 53 mM and 131 ? 117 mM respectively.Cl^– balanced the sum of K⁺ Na⁺. Influxes of ²²Na⁺, ⁴²K⁺,and ³⁶C1^– were measured in cells in which the ions werein the steady state. Averages of 610 and 6.6 nmol m^–2s^–1 were obtained for Na⁺ and K⁺ respectively. Cl^–influx was divided into 2 phases with values of 1540 and 178mmol m^–2 s^–1. The faster influx was considered tobe across the outer cell membrane. The membrane responsiblefor the slower influx has not been identified. By comparingvalues of the potential difference calculated from the Nernstand Goldman equations, it was concluded that Na⁺ and K⁺ areprobably controlled by active mechanisms, whereas cell Cl^–is likely to be at thermodynamic equilibrium with the medium.     

Ion Fluxes to the Vacuole of Nitella translucens

The time course of the appearance in the vacuole of Nitellatranslucens and of Tolypella intricata of tracer from the outsidesolution has been studied over short periods of uptake. Thereare two components of chloride transfer to the vacuole, a fastcomponent linear with time and a second component at longertimes whose behaviour is reasonably well described in termsof a single rate constant for exchange; a constant fractionof the total entry is in the fast component and the apparentrate constant for the second component is proportional to theinflux. In Nitella the path of rapid transfer involves chlorideand sodium, and may also involve a small but variable amountof potassium, but in Tolypella potassium has a significant componentof rapid transfer; these correspond to the cations for whichchloride-linked components of cation influx have been shownby another worker. Over both parts of the time course the level of activity inthe cytoplasm specifies, not the rate of transfer to the vacuoleas would be expected, but the rate as a fraction of the influx;the processes of influx to the cell and transfer to the vacuoleare intimately linked. It is difficult to explain the results in terms of static membranesand fixed compartments. An explanation in terms of the sequence,entry of salt by pino-cytotic vesicles at the plasmalemma, fusionof these vesicles with the endoplasmic reticulum after someloss of tracer to the surrounding cytoplasm, and transfer tothe vacuole in minivacuoles formed from the endoplasmic reticulum,is consistent with the time course found. A model of this kind,involving transport by a dynamic membrane system, seems necessaryto explain the results.     

Transient-State Analysis of Metabolic Fluxes in Crabtree-Positive and Crabtree-Negative Yeasts

In bakers' yeast, an immediate alcoholic fermentation begins when a glucose pulse is added to glucose-limited, aerobically grown cells. The mechanism of this short-term Crabtree effect was investigated via a comparative enzymic analysis of eight yeast species. It was established that the fermentation rate of the organisms upon transition from glucose limitation to glucose excess is positively correlated with the level of pyruvate decarboxylase (EC 4.1.1.1). In the Crabtree-negative yeasts, the pyruvate decarboxylase activity was low and did not increase when excess glucose was added. In contrast, in the Crabtree-positive yeasts, the activity of this enzyme was on the average sixfold higher and increased after exposure to glucose excess. In Crabtree-negative species, relatively high activities of acetaldehyde dehydrogenases (EC 1.2.1.4 and EC 1.2.1.5) and acetyl coenzyme A synthetase (EC 6.2.1.1), in addition to low pyruvate decarboxylase activities, were present. Thus, in these yeasts, acetaldehyde can be effectively oxidized via a bypass that circumvents the reduction of acetaldehyde to ethanol. Growth rates of most Crabtree-positive yeasts did not increase upon transition from glucose limitation to glucose excess. In contrast, the Crabtree-negative yeasts exhibited enhanced rates of biomass production which in most cases could be ascribed to the intracellular accumulation of reserve carbohydrates. Generally, the glucose consumption rate after a glucose pulse was higher in the Crabtree-positive yeasts than in the Crabtree-negative yeasts. However, the respiratory capacities of steady-state cultures of Crabtree-positive yeasts were not significantly different from those of Crabtree-negative yeasts. Thus, a limited respiratory capacity is not the primary cause of the Crabtree effect in yeasts. Instead, the difference between Crabtree-positive and Crabtree-negative yeasts is attributed to differences in the kinetics of glucose uptake, synthesis of reserve carbohydrates, and pyruvate metabolism.     

Estimating Metabolic Fluxes Using a Maximum Network Flexibility Paradigm

MotivationGenome-scale metabolic networks can be modeled in a constraint-based fashion. Reaction stoichiometry combined with flux capacity constraints determine the space of allowable reaction rates. This space is often large and a central challenge in metabolic modeling is finding the biologically most relevant flux distributions. A widely used method is flux balance analysis (FBA), which optimizes a biologically relevant objective such as growth or ATP production. Although FBA has proven to be highly useful for predicting growth and byproduct secretion, it cannot predict the intracellular fluxes under all environmental conditions. Therefore, alternative strategies have been developed to select flux distributions that are in agreement with experimental “omics” data, or by incorporating experimental flux measurements. The latter, unfortunately can only be applied to a limited set of reactions and is currently not feasible at the genome-scale. On the other hand, it has been observed that micro-organisms favor a suboptimal growth rate, possibly in exchange for a more “flexible” metabolic network. Instead of dedicating the internal network state to an optimal growth rate in one condition, a suboptimal growth rate is used, that allows for an easier switch to other nutrient sources. A small decrease in growth rate is exchanged for a relatively large gain in metabolic capability to adapt to changing environmental conditions.ResultsHere, we propose Maximum Metabolic Flexibility (MMF) a computational method that utilizes this observation to find the most probable intracellular flux distributions. By mapping measured flux data from central metabolism to the genome-scale models of Escherichia coli and Saccharomyces cerevisiae we show that i) indeed, most of the measured fluxes agree with a high adaptability of the network, ii) this result can be used to further reduce the space of feasible solutions iii) this reduced space improves the quantitative predictions made by FBA and contains a significantly larger fraction of the measured fluxes compared to the flux space that was reduced by a uniform sampling approach and iv) MMF can be used to select reactions in the network that contribute most to the steady-state flux space. Constraining the selected reactions improves the quantitative predictions of FBA considerably more than adding an equal amount of flux constraints, selected using a more naïve approach. Our method can be applied to any cell type without requiring prior information.AvailabilityMMF is freely available as a MATLAB plugin at: http://cs.ru.nl/~wmegchel/mmf.     

Effect of Sudden Salt Stress on Ion Fluxes in Intact Wheat Suspension Cells

Although salinity is one of the major problems limiting agriculturalproduction around the world, the underlying mechanisms of highNaCl perception and tolerance are still poorly understood. Theeffects of different bathing solutions and fusicoccin (FC),a known activator of plasma membrane ATPase, on plasma membranepotential (E_m) and net fluxes of Na⁺, K⁺and H⁺were studied inwheat suspension cells (Triticum aestivum) in response to differentNaCl treatments. E_mof cells in Murashige and Skoog (MS) mediumwas less negative than in cells exposed to a medium containing10 mM KCl + 0.1 m M CaCl₂(KSM) and to a basic salt medium (BSM),containing 1 m M KCl and 0.1 m M CaCl₂. Multiphasic Na⁺accumulationin cells was observed, peaking at 13 min after addition of 120m M NaCl to MS medium. This time scale was in good agreementwith net Na⁺flux changes measured non-invasively by moving ion-selectivemicroelectrodes (the MIFE system). When 120 m M NaCl was addedto all media studied, a quick rise of Na⁺influx was reversedwithin the first 20 min. In both 120 and 20 m M NaCl treatmentsin MS medium, net Na⁺efflux was observed, indicating that activeNa⁺transporters function in the plant cell response to saltstress. Lower external K⁺concentrations (KSM and BSM) and FCpre-treatment caused shifts in Na⁺fluxes towards net influxat 120 m M NaCl stress. Copyright 2000 Annals of Botany Company Sodium, potassium, proton, membrane potential, fusicoccin, salt stress, wheat, Triticum aestivum     

Ion Fluxes in 'Isolated' Guard Cells of Commelina communis L.

Ion fluxes have been measured in ‘isolated’ guardcells of Commelina communis L. using ⁸⁶RbCl and K⁸²Br, in epidermalstrips in which all cells other than guard cells have been killedby treatment at low pH. To avoid problems of slow free spaceexchange most fluxes have been measured at pH 3.9, at whichstomata open well in K(Rb) Cl(Br) and are stable for many hours.At pH 3.9 the intracellular ⁸⁶Rb exchanged as a single compartmentwith a half-time of 2–3 h, independent of external concentration(C_o). The influx of ⁸⁶Rb rose with concentration, to a V_maxof about 23 pmol mm^–2 h^–1. The efflux curve of ⁸²Brcould be well fitted by two exponential terms, with half-timesof 38 min (independent of C_o), and 5–35 h (falling withincreasing C_o). Bromide contents of cytoplasm and vacuole (Q_cand Q_v), and fluxes at plasmalemma and tonoplast, were calculatedfrom the efflux kinetics. Over C_o 20–60 mM, as the apertureincreased from 7 µm to 17 µm, the tonoplast flux(0.5–11.5 pmol mm^–2h^–1) was always much lessthan the plasmalemma flux (7–77 pmol mm^–2 h^–1).Q_c and Q_v both increased with aperture. The increase in Q_c of10.3 pmol mm^–2 µm^–1 is adequate to accountfor the osmotic changes required to change the aperture, aspreviously estimated. However, the change in vacuolar contentof only 5.9 pmol mm^–2 µm^–1 is much too smallto account for the osmotic changes required, or to balance thecytoplasmic changes. It appears therefore that increasing KBroutside not only increases the cytoplasmic salt content, andthe Br flux at the tonoplast, but also stimulates the vacuolaraccumulation of some other solute.     

Quantitation of Rates of Transport, Metabolic Fluxes, and Cytoplasmic Levels of Inorganic Carbon in Maize Root Tips during K Ion Uptake

Our aim was to determine whether fixation of inorganic carbon (C_i), due to phosphoenolpyruvate carboxylase activity, is limited by the availability of C_i in the cytoplasm of maize (Zea mays L.) root tips. Rates of C_i uptake and metabolism were measured during K₂SO₄ treatment, which stimulates dark C_i fixation. ¹³C_i uptake was followed by ¹³C-nuclear magnetic resonance (NMR); 5 millimolar K₂SO₄ had no significant effect on ¹³C_i influx. The contribution of respiratory CO₂ production to cytoplasmic HCO₃⁻ was measured using in vivo ¹³C-NMR and ¹H-NMR of cell extracts; K₂SO₄ treatment had no effect on respiratory CO₂ production. The concentration of cytoplasmic HCO₃⁻ was estimated to be approximately 11 millimolar, again with K₂SO₄ having no significant effect. These experiments allowed us to determine the extent to which extracellularly supplied ¹⁴C_i was diluted in the cytoplasm by respiratory CO₂ and thereby measure phosphoenolpyruvate (PEP) carboxylase activity in vivo using ¹⁴C_i. PEP carboxylase activity in root tips was enhanced approximately 70% over controls within 12 minutes of the addition of 5 millimolar K₂SO₄. The activity of carbonic anhydrase, which provides PEP carboxylase with C_i, was determined by saturation transfer ¹³C-NMR to be more than 200 times that of PEP carboxylase in vivo. The regulation of PEP carboxylase in K₂SO₄-treated roots is discussed.     

一种中间代谢途径代谢通量的定量分析方法

以¹³C标记的碳源,用二维核磁共振技术(¹H-¹³C,HMQC)测定代谢中产生的氨基酸标记模式,研究对中间代谢途径胞内代谢通量进行定量分析的方法.通过开发软件包,改进同位素分布的数学模型,提出了反应映射矩阵(RMM)等概念.由简化算法,提高程序的执行效率,建立了定量分析胞内代谢通量的平台.代谢模型涉及了糖酵解途径、磷酸戊糖途径、三羧酸循环、几种回补反应、发酵途径和氨基酸合成途径.     

Association between Ion Fluxes and Defense Responses in Pea and Cowpea Tissues

The glycopeptide elicitor from a pea pathogen, Mycos-phaerellapinodes, induced rapid alkalinization and increases in levelsof Na² and K² ions in the extracellular solution upon contactwith pea and cowpea tissues. The presence of monensin, nigericin,lidocaine, quinidine or phenytoin together with the elicitormarkedly inhibited these changes, whereas the presence of valinomycin,gramicidin D, tetraethylammonium, CsCl and aminopyridine didnot. The production of phytoalexins in pea and cowpea tissueswas also strongly inhibited by the simultaneous presence ofthe former reagents but not of the latter reagents. Inhibitoryeffects on the production of phytoalexins were diminished whenmonensin, nigericin or a Na²-channel blocker was applied 3 hafter the start of treatment with elicitor. Furthermore, orthovanadateand neomycin, which suppress defense responses in both tissues,also inhibited the above mentioned changes. By contrast, thespecies-specific suppressor from M. pinodes inhibited the elicitor-in-ducedrelease of Na² and K² ions from pea tissues, but, conversely,by itself it elicited either the defense response or the releaseof Na² and K² ions from cowpea tissues. The results indicatethat these ion-related changes, in particular the efflux ofNa² and K² ions, might be closely associated with the signaltransduction system for defense responses at the tissue level. (Received March 21, 1996; Accepted March 27, 1997)