Self-referencing optrodes for measuring spatially resolved, real-time metabolic oxygen flux in plant systems

The ability to non-invasively measure metabolic oxygen flux is a very important tool for physiologists interested in a variety of questions ranging from basic metabolism, growth/development, and stress adaptation. Technologies for measuring oxygen concentration near the surface of cells/tissues include electrochemical and optical techniques. A wealth of knowledge was gained using these tools for quantifying real-time physiology. Fiber-optic microprobes (optrodes) have recently been developed for measuring oxygen in a variety of biomedical and environmental applications. We have adopted the use of these optical microsensors for plant physiology applications, and used the microsensors in an advanced sensing modality known as self-referencing. Self-referencing is a non-invasive microsensor technique used for measuring real-time flux of analytes. This paper demonstrates the use of optical microsensors for non-invasively measuring rhizosphere oxygen flux associated with respiration in plant roots, as well as boundary layer oxygen flux in phytoplankton mats. Highly sensitive/selective optrodes had little to no hysteresis/calibration drift during experimentation, and an extremely high signal-to-noise ratio. We have used this new tool to compare various aspects of rhizosphere oxygen flux for roots of Glycine max, Zea mays, and Phaseolus vulgaris, and also mapped developmentally relevant profiles and distinct temporal patterns. We also characterized real-time respiratory patterns during inhibition of cytochrome and alternative oxidase pathways via pharmacology. Boundary layer oxygen flux was also measured for a phytoplankton mat during dark:light cycling and exposure to pharamacological inhibitors. This highly sensitive technology enables non-invasive study of oxygen transport in plant systems under physiologically relevant conditions.     

Simultaneous minute by minute determination of unidirectional and net water fluxes in frog urinary bladder A reexamination of the two barriers in series hypothesis

Unidirectional and net water fluxes were simultaneously estimated in frog urinary bladder. The minute by minute tritiated water (³HOH) transepithelial flux and the net volume of fluid traversing the tissue were employed. It was observed that: (1) the time course of the increase in the ³HOH flux induced by antidiuretic hormone had a very similar pattern to that reported for the increase in the net movement. (2) Unstirred layers strongly affected the magnitude of the antidiuretic hormone-induced increase in ³HOH fluxes while the time course of the response was almost non-affected. In non-stimulated bladders ³HOH fluxes were poorly modified by medium stirring. New steadystate conditions for ³HOH fluxes were established 1 min after stirring rate modifications. (3) The simultaneously determined net water flux was not affected by a modification in the unstirred layers, indicating that the variations in the measured net water fluxes are a good estimation of the changes in the mucosal border permeability. (4) The presence of an osmotic gradient during hormonal challenge (implying net water fluxes, cell swelling and dilation of the intracellular spaces) did not modify the time course of ³HOH movements. These results suggest that the time course of the increase in water permeability is an intrinsic characteristic of the experimental system that could result from the addition of permeability units that increase in number during the development of the harmonal action.     

Calculation of poloidal velocity in the tokamak plasma with allowance for density inhomogeneity and diamagnetic drift of ions

A one-dimensional evolution equation for the angle-averaged poloidal momentum of the tokamak plasma is derived in the framework of reduced magnetohydrodynamics with allowance for density inhomogeneity and diamagnetic drift of ions. In addition to fluctuations of the E × B drift velocity, the resulting turbulent Reynolds stress tensor includes fluctuations of the ion density and ion pressure, as well as turbulent radial fluxes of particles and heat. It is demonstrated numerically by using a particular example that the poloidal velocity calculated using the refined one-dimensional evolution equation differs substantially from that provided by the simplified model. When passing to the new model, both the turbulent Reynolds force and the Stringer-Winsor force increase, which leads to an increase in the amplitude of the ion poloidal velocity. This, in turn, leads to a decrease in turbulent fluxes of particles and heat due to the effect of shear decorrelation.     

The relative merits of open- and closed-path analysers for measurement of eddy fluxes

Theoretical and practical aspects of measuring eddy fluxes of trace gases using open-and closed-path analysers are presented. Trace gas fluxes measured with an open-path analyser require the concurrent measurement of sensible and latent heat fluxes to correct for density fluctuations in trace gas concentration caused by these fluxes. A closed-path analyser eliminates the corrections due to sensible heat flux, but not for water vapour, provided temperature fluctuations are completely removed without significantly reducing fluctuations in the trace gas mixing ratio. Theory for the design of heat exchangers and for the attenuation of concentration fluctuations during air flow through tubes is used to provide design criteria for closed-path systems. Spectral transfer functions are used to estimate flux losses caused by flow through the sampling tube and gas analyser. Other factors considered include cross-sensitivity of infrared CO₂ analysers to water vapour, and deterioration of system performance caused by contaminants on the walls of sampling tubes. Of two open-path, infrared CO₂ analysers tested, one showed a strong interaction between CO₂ and water vapour, while the other showed little sensitivity to the presence of water vapour, other than caused by dilution. A commercial closed-path CO₂ analyser also showed little cross-sensitivity to water vapour. Compared to results for a clean sampling tube, the spectral bandwidth for water vapour fluctuations decreased significantly after several weeks of sampling. No such deterioration in bandwidth was observed for CO₂. These findings are attributed to differential adsorption/desorption of water vapour by dust or salt on the tubing walls. Rain and dust must be removed from open-path analysers to obtain satisfactory measurements. Careful system design and maintenance is required for both open- and closed-path systems to ensure satisfactory long-term measurement of trace gas fluxes. With these precautions, both approaches will provide satisfactory flux measurements.     

北京城郊地区二氧化碳通量特征

利用位于北京市顺义气象局45 m气象塔上36 m高度的湍流观测资料,对该区域2008年11月1日至2009年10月31日共365d的二氧化碳通量(CO2)的时间变化和各方位的分布特征进行了分析研究,并计算了CO2年排放量。结果表明,CO2受交通因素和居民日常生活排放的影响较小,冬季耗能取暖会显著增加CO2的排放量;受供暖排放和植物生长季节光合作用的影响,冬季的CO2通量值在全天绝大多数时刻均高于其他季节,其日平均值为15.6μmol m-2s-1,显著高于春、夏、秋季的日平均值5.6、5.7和8.8μmol m-2s-1(t-test,P0.001)。各方向CO2通量值的大小与其源区内土地利用/覆盖方式以及建筑物的使用功能和使用性质密切相关,住宅楼、饭店、工厂、旅馆等人工建筑面积占比例越大,CO2排放量越大;而植被覆盖比例较高的方向CO2值较小。观测点周边区域是CO2的排放源,且年平均排放量达到13.6 kg m-2a-1,但低于同一时期北京市内高密度住宅区域的CO2年排放量。     

EPR study of electron transport in the cyanobacterium Synechocystis sp. PCC 6803: Oxygen-dependent interrelations between photosynthetic and respiratory electron transport chains

In this work, we investigated electron transport processes in the cyanobacterium Synechocystis sp. PCC 6803, with a special emphasis focused on oxygen-dependent interrelations between photosynthetic and respiratory electron transport chains. Redox transients of the photosystem I primary donor P700 and oxygen exchange processes were measured by the EPR method under the same experimental conditions. To discriminate between the factors controlling electron flow through photosynthetic and respiratory electron transport chains, we compared the P700 redox transients and oxygen exchange processes in wild type cells and mutants with impaired photosystem II and terminal oxidases (CtaI, CydAB, CtaDEII). It was shown that the rates of electron flow through both photosynthetic and respiratory electron transport chains strongly depended on the transmembrane proton gradient and oxygen concentration in cell suspension. Electron transport through photosystem I was controlled by two main mechanisms: (i) oxygen-dependent acceleration of electron transfer from photosystem I to NADP⁺, and (ii) slowing down of electron flow between photosystem II and photosystem I governed by the intrathylakoid pH. Inhibitor analysis of P700 redox transients led us to the conclusion that electron fluxes from dehydrogenases and from cyclic electron transport pathway comprise 20-30% of the total electron flux from the intersystem electron transport chain to P700⁺.     

Single-cell, real-time measurements of extracellular oxygen and proton fluxes fromSpirogyra grevilleana

Summary We have adapted the self-referencing microelectrode technique to allow sensitive and noninvasive measurement of oxygen fluxes around single cells. The self-referencing technique is based on the translational movement of a selective microelectrode through the gradient next to the cell wall or membrane. The electrode is moved at a known frequency and between known points. The differential electrode output values are converted into a directional measurement of flux by the Fick equation. By coupling the newly developed oxygen-selective self-referencing electrochemical microelectrode (SREM-O₂) system with self-referencing ionselective proton measurements (SRIS-H⁺) we have characterized oxygen and proton fluxes from a single cell of the filamentous green algaSpirogyra gre illeana (Hass.). Oxygen showed a net efflux and protons showed a net influx when the cell was illuminated. These photosynthesis-dependent fluxes were found to be spatially associated with the chloroplasts and were sensitive to treatment with dichlorophenyldimethylurea. In the dark the directions of oxygen and proton fluxes were reversed. This oxygen influx was associated with mitochondrial respiration and was reduced by 78% when the cells was treated with 0.5 mM KCN. The residual cyanide-resistant respiration was inhibited by the application of 5 mM salicylhydroxamic acid, an inhibitor of the alternative oxidase. Similarly the cytochrome pathway was also inhibited by the presence of 20 M NO, while the cyanide-resistant alternative oxidase was not. These results demonstrate the use of the newly developed SREM-O₂ system to measure and characterize metabolic fluxes at a level of sensitivity that allows for subcellular resolution. These measurements, in conjunction with SERIS-H⁺ measurements, have led to new insights in our understanding of basic cellular physiology in plant cells.Abbreviations SRIS self-referencing ion selective - SREM self-referencing electrochemical microelectrode - ICP inductive coupled plasma spectroscopy     

Molecule capture by olfactory antennules: Mantis shrimp

 A critical step in the process of olfaction is the movement of odorant molecules from the environment to the surface of a chemosensory structure. Many marine crustaceans capture odorant molecules with arrays of chemosensory sensilla (aesthetascs) on antennules that they flick through the water. We developed a model to calculate molecule flux to the surfaces of aesthetascs in order to study how the size, aesthetasc spacing, and flick kinematics of olfactory antennules affect their performance in capturing molecules from the surrounding water. Since the three-dimensional geometry of an aesthetasc-bearing antennule is complex, dynamically-scaled physical models can often provide an efficient method of determining the fluid velocity field through the array. Here we present a method to optimize the incorporation of such measured velocity vector fields into a numerical simulation of the advection and diffusion of odorants to aesthetasc surfaces. Furthermore, unlike earlier models of odorant interception by antennae, our model incorporates odorant concentration distributions that have been measured in turbulent ambient flows. By applying our model to the example of the olfactory antennules of mantis shrimp, we learned that flicking velocity can have profound effects on odorant flux to the aesthetascs if they operate in the speed range in which the leakiness of the gaps between the aesthetascs to fluid movement is sensitive to velocity. This sensitivity creates an asymmetry in molecule fluxes between outstroke and return stroke, which results in an antennule taking discrete samples in space and time, i.e. “sniffing”. As stomatopods grow and their aesthetasc Reynolds number increases, the aesthetasc arrangement on the antennule changes in a way that maintains these asymmetries in leakiness and molecule flux between the outstroke and return stroke, allowing the individual to continue to take discrete samples as it develops. Received: 24 May 2000 / Revised version: 8 May 2001 / Published online: 7 December 2001     

南京地区大口径闪烁仪与涡动相关仪感热通量观测对比

湍流是地表与大气间物质与能量交换的主要形式,因而准确观测湍流通量历来是城市边界层研究的重要问题。本研究基于架设在南京信息工程大学内的大口径闪烁仪(large aperture scintillometer,LAS)和涡动相关仪(eddy covariance,EC)的同步观测,对比了LAS测得的感热通量和EC测得的感热通量的差异,结合归一化植被指数(NDVI)和归一化建筑指数(NDBI),分析了下垫面不均匀性对于两种仪器测得感热通量的影响。结果表明:城市地区LAS与EC具有较好的相关性(R²=0.76),拟合线斜率为0.95;白天,LAS的感热通量大于EC的感热通量,二者差值为18.8～39.4 W·m^-2;夜间,二者均在零值附近波动,差值为4.8～28.7 W·m^-2;月尺度上两种仪器的差值8月最大,其次为7月、4月,6月最小;差异产生的主要原因是风向造成的通量源区不同;通量源区内的NDVI值越大,感热通量与净辐射之比越小,二者呈显著负相关(k=-0.34,P<0.05);NDBI值越大,感热通量与净辐射之比越大...     

区域尺度无人机涡动相关通量观测系统的应用研究

陆地生态系统的水热循环与碳循环是陆地表层系统中物质能量循环的核心,其中区域尺度地表水、热、碳通量的直接观测是当下陆地生态系统通量观测与模拟研究中的热点与难点。机载涡动相关方法能够直接观测区域尺度生态系统通量,基于无人机平台的涡动相关通量观测技术同时兼具了区域覆盖性与经济灵活性等优点,是机载通量观测技术的最新发展方向。在介绍机载涡动相关通量观测方法的主要技术原理、观测特点以及无人机通量观测系统组成的基础上,通过在相对均匀的区域开展无人机与地面通量观测对比试验,采用谱分析、观测结果对比以及源区分析等方式对无人机通量观测系统的性能进行了初步评价。结果表明：无人机通量观测系统能够实现对大气高频湍流信号的有效采样;无人机与地面观测的湍流通量具有较好的一致性,但是感热和CO₂通量出现了低估、潜热和摩擦风速出现了高估;观测平台与仪器的差异、垂直通量辐散、大气边界层条件、不同的地面源区及地表异质性的影响是造成二者差异的潜在主要因素。最后对未来研究目标进行了展望,以进一步推动该技术在相关领域中的应用。     

Metabolic flux and robustness analysis of glycerol metabolism in <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

The knowledge of the mechanism of flux distribution will benefit understanding cell physiology and regulation of metabolism. In this study, the measured fluxes obtained under steady-state conditions were used to estimate intracellular fluxes and identify the robustness of branch points of the anaerobic glycerol metabolism in Klebsiella pneumoniae for the production of 1,3-propanediol by metabolic flux analysis. The biomass concentration increased as NADH₂/NAD⁺ decreased at low initial concentration and inversed at high initial glycerol concentration. The flux distribution revealed that the branch points of glycerol and dihydroxyacetonephosphate were rigid to the environmental conditions. However, the pyruvate and acetyl coenzyme A metabolisms gave cells the flexibility to regulate the energy and intermediate fluxes under various environmental conditions. Additionly, it was found that the formation rate of ethanol and the ratio of pyruvate dehydrogenase to pyruvate formate lyase appeared visible fluctuations at high glycerol uptake rate.     

Estimation of turbulent shear stresses in pulsatile flow immediately downstream of two artificial aortic valves in vitro

Measuring turbulent shear stresses is of major importance in artificial heart valve evaluation. Bi- and unidirectional fluid velocity measurements enable calculation of Reynolds shear stress ( ) and Reynolds normal stress ( ). τ is important due to the relation to hemolysis and thrombus formation, but σ is the only obtainable parameter in vivo. Therefore, determination of a correlation factor between τ and σ is pertinent.

In a pulsatile flow model, laser Doppler (LDA) and hot-film (HFA) anemometry were used for simultaneous bi- and unidirectional fluid velocity measurements downstream of a Hall Kaster and a Hancock Porcine aortic valve. Velocities were registered in two flow field locations and at four cardiac outputs. The velocity signals were subjected to analog signal processing prior to digital turbulence analysis, as a basis for calculation of τ and σ.

A correlation factor of 0.5 with a correlation coefficient of 0.97 was found between the maximum Reynolds shear stress and Reynolds normal stress, implying . In vitro estimation of turbulent shear stresses downstream of artificial aortic valves, based on the axial velocity component alone, seems possible.     

Contribution of the alternative pathway to respiration during thermogenesis in flowers of the sacred lotus

We report results from in vivo measurements, using oxygen isotope discrimination techniques, of fluxes through the alternative and cytochrome respiratory pathways in thermogenic plant tissue, the floral receptacle of the sacred lotus (Nelumbo nucifera). Fluxes through both pathways were measured in thermoregulating flowers undergoing varying degrees of thermogenesis in response to ambient temperature. Significant increases in alternative pathway flux were found in lotus receptacles with temperatures 16 degrees C to 20 degrees C above ambient, but not in those with lesser amounts of heating. Alternative pathway flux in the hottest receptacles was 75% of the total respiratory flux. In contrast, fluxes through the cytochrome pathway did not change significantly during thermogenesis. These data support the hypothesis that increased flux through the alternative pathway is responsible for heating in the lotus and that it is unlikely that uncoupling proteins, which would have produced increased fluxes through the cytochrome pathway, contribute significantly to heating in this tissue. Comparisons of actual flux, with capacity determined using inhibitors, suggested that the alternative pathway was operating at close to maximum capacity in heating tissues of lotus. However, in nonheating tissues the inhibitor data significantly overestimated the alternative pathway flux. This confirms that isotopic measurements are necessary for accurate determination of fluxes through the two pathways.     

Characterization of benthic microhabitat: An experimental system for aquatic insects

An experimental system used to determine microhabitat current velocity and microhabitat selection by aquatic insects is described. The experimental system includes a microvelocity probe and a hydraulically calibrated artificial substrate. A thermistor velocity probe detects flow velocities to 0.5 m s^–1 near the surface of substrates at locations inhabited by aquatic insects. The artificial substrate was designed to provide two major habitat types, highly turbulent vortex areas and regions with unidirectional, near laminar flow. Substrate calibration and microhabitat characteristics of the substrates are demonstrated. Experimental studies of Simulium sp. location on substrates indicated that while simuliid larvae are characteristic of lotic, erosional habitats, actual microhabitats selected are governed by substantially lower flow velocity.     

Soil-surface CO<Superscript>2</Superscript> fluxes in a <Emphasis Type="Italic">Deyeuxia angustifolia</Emphasis> wetland in Sanjiang Plain,China

In many temperate-zone ecosystems, seasonal changes in environmental and biological factors influence the dynamics and magnitude of surface–atmosphere exchange. Research was conducted between July and October 2001 to measure growing season surface-layer fluxes of CO₂ in a Deyeuxia angustifolia dominated wetland on the Sanjiang Plain in northeastern China. Seasonal fluctuation and daily change in soil-surface CO₂ fluxes were measured as well as the edaphic factors controlling CO₂ fluxes. Soil-surface CO₂ fluxes were measured with a closed-chamber system. The results revealed that there were both seasonal fluctuations and daily change in CO₂ fluxes. The ranges of measured soil-surface CO₂ flux were 0.208 – 1.265 g CO₂m^–2h^–1. Soil-surface CO₂ fluxes averaged 0.620 g CO₂ m^–2h^–1. An analysis of several edaphic factors including soil temperature and soil moisture of the D. angustifolia wetland showed that there was a significant relationship between flux and temperature (R² = 0.77).     

A model investigation of the velocity and pressure spectra in vascular murmurs

A physical model consisting of an axisymmetrical jet in a rigid plexiglass pipe was used to study the flow and pressure fluctuations downstream from an aortic stenosis. The fluctuating velocity components, u and v, at several locations in the steady liquid jet were measured using a laser Doppler anemometer system. Simultaneous wall pressure fluctuations were monitored by an array of nine miniature pressure transducers wall mounted in the axial direction. This paper presents the detailed measurements of mean velocity profiles, turbulent intensity distributions and RMS pressure fluctuations. The energy spectra obtained for the pressure fluctuations and the u and v velocity components are compared. Contrary to earlier works, we found that the differences between peak frequencies of the pressure spectra and the characteristic frequencies of the velocity spectra vary with positions downstream from the nozzle. These differences are discussed in light of pseudosound generation by the eddy structures in the stenotic flow field.     

Cartesian-diver microrespirometry: a technique for the accurate measurement of respiratory flux in plant cells

Abstract. A Cartesian-diver microrespirometer system is described which can be used to measure respiratory fluxes of oxygen accurately for cells of higher plants in a liquid phase. This microrespirometry technique has been adapted from protozoological and microfaunal studies to plant physiology. The Cartesian-diver has considerable scope for investigation of oxygen flux in plant cells and has several advantages compared to the oxygen electrode in terms of sensitivity to changing oxygen levels in respiring material. Because the volumes of liquid are small in the Cartesian-divers, diffusional distances arc measured in micrometres and there is no need for stirring to overcome diffusional problems, thus minimizing the risk of mechanical damage to the experimental material. In addition, only very small quantities of experimental material are required for the Cartesian-diver which is invaluable where only limited amounts of tissue or numbers of cells can be obtained. Examples of respiratory oxygen consumption by protoplasts from intercalary meristematic regions of light-grown barley ( Hordeum vulgare L.c.v. Patty) seedlings, in response to abscisic and gibberellic acids, are presented. The advantages and disadvantages of Cartesian-diver microrespirometry compared to oxygen electrodes are also discussed.     

Estimation of turbulent shear stresses in pulsatile flow immediately downstream of two artificial aortic valves in vitro

Measuring turbulent shear stresses is of major importance in artificial heart valve evaluation. Bi- and unidirectional fluid velocity measurements enable calculation of Reynolds shear stress ( ) and Reynolds normal stress ( ). τ is important due to the relation to hemolysis and thrombus formation, but σ is the only obtainable parameter in vivo. Therefore, determination of a correlation factor between τ and σ is pertinent.In a pulsatile flow model, laser Doppler (LDA) and hot-film (HFA) anemometry were used for simultaneous bi- and unidirectional fluid velocity measurements downstream of a Hall Kaster and a Hancock Porcine aortic valve. Velocities were registered in two flow field locations and at four cardiac outputs. The velocity signals were subjected to analog signal processing prior to digital turbulence analysis, as a basis for calculation of τ and σ.A correlation factor of 0.5 with a correlation coefficient of 0.97 was found between the maximum Reynolds shear stress and Reynolds normal stress, implying . In vitro estimation of turbulent shear stresses downstream of artificial aortic valves, based on the axial velocity component alone, seems possible.     

LAS测算森林冠层上方温度结构参数的可行性

利用位于河南省济源市的华北低丘山地30年生栓皮栎-侧柏-刺槐人工混交林2010年4月至8月每月连续7d,LAS直接测算的森林冠层上方湍流结构参数,与经过湍流谱方法计算处理的三维超声风速/温度仪的观测数据比较,分析LAS测算低丘山地森林冠层温度湍流结构的可行性。结果表明:水平风速和温度湍流谱都有明显的惯性区出现(斜率-2/3);LAS直接测算的湍流温度结构参数与利用该惯性区的数据计算的结果具有较好的一致性,说明在起伏非均匀下垫面上,采用LAS观测湍流结构的变化情况具有较好的可行性。     

Comparison of CO<Subscript>2</Subscript> and H<Subscript>2</Subscript>O fluxes over grassland vegetations measured by the eddy-covariance technique and by open system chamber

Measurements of CO₂ and H₂O fluxes were carried out using two different techniques—eddy-covariance (EC) and open system gas exchange chamber (OC)—during two-years’ period (2003–2004) at three different grassland sites. OC measurements were made during fourteen measurement campaigns. We found good agreement between the OC and EC CO₂ flux values (n = 63, r ² = 0.5323, OC F_CO2 = −0.6408+0.9508 EC F_CO2). The OC F_H2O values were consistently lower than those measured by the EC technique, probably caused by the air stream difference inside and outside the chamber. Adjusting flow rate within the chamber to the natural conditions would be necessary in future OC measurements. In comparison with EC, the OC proved to be a good tool for gas exchange measurements in grassland ecosystems.