植物ms级叶绿素荧光动力学数据采集和分析软件

本文介绍了一套可在ＩＢＭＰＣ（兼容）微机上运行的植物ｍｓ级荧光动力学数据采集和分析软件。本软件用Ｃ语言写成,具有汉化界面、菜单驱动、彩色人机对话窗口、操作简便、灵活,兼容性好等优点。本文着重讨论了本软件如何减少荧光动力学测量中的误差、在不过分占用内存的情况下扩大采样时间范围以及如何精确测量固定荧光（Ｆｏ）和偏转荧光（Ｆｉ）等问题。本软件和我们自行组装的植物ｍｓ级动力学荧光计———植物产量荧光计ＰＦＭ１０１型可广泛应用于农业、植物生态学及植物生理学的光合测量和研究     

适用于细胞荧光测量的多道显微荧光计

建立了由倒置荧光显微镜和光学多道分析仪(OMA)连接而组成的适用于细胞荧光测量的多道显微荧光计,编制了数据处理程序。利用这一装置测量了单个细胞,多细胞的荧光光谱和拓扑(topography)。和传统的显微荧光计相比,该装置具有测量灵敏度和精度高、速度快等特点,可用来进行活细胞动态过程的研究。     

一种新型植物动力学荧光仪

当植物从暗中转到光下时 ,其体内叶绿素 (Ｃｈｌ)荧光强度会随照光时间产生有规律的变化 ,这就是植物荧光诱导现象。由于它能够灵敏、快速、简便和无损伤地探测植物体内光合生理状况及环境因子对植物的影响 ,近年来在植物生理、植物生态、农业、林业、环境污染和遥感等领域得到重视和应用［１,２,３］。植物动力学荧光仪有调制式和非调制式两类 ,非调制式荧光仪特别适合于荧光诱导上升曲线及曲线中偏转荧光 (ＦＩ)和荧光上升互补面积 (ＣＡ)的研究 ,其中ＦＩ 是快速简便地探测体内ＰＳＩＩ无活性中心相对含量的重要途径 ,后者与光合激发能…     

鲫鱼视网膜双极细胞上GABAC和GABAA受体的动力学差异

用全细胞模式的膜片钳技术研究了鲫鱼视网膜双极细胞上的GABAC和GABAA受体激活(activation)、失敏(desensitization)和失活(deactivation)等动力学特性.研究表明GABAC受体的反应动力学一般慢于GABAA受体.两者的激活动力学可用单指数函数很好拟合,GABAC受体的拟合时间常数(τ)为44.57 ms,而GABAA受体的平均时间常数是10.86 ms.再者,GABAA受体的失敏具有快、慢两个成分,其时间常数分别是ιfast=2.16 s和τslow=19.78 s,而GABAC的失敏则遵从单指数规律,时间常数为6.98 s.两种受体的失活均能为双指数函数拟合,其中GABAC受体的时间常数(ιfast=674.8 ms,τslow=2090ms)远大于GABAA受体的(ιfast=42.07 ms,ιslow=275.1 ms).这些动力学特性的差异提示,GABAC和GABAA受体可能在不同的频域参与视网膜的信号处理.     

瞬时与延迟叶绿素荧光及820nm光反射动力学同步测量技术在光合作用研究中的应用

在光合作用研究中,通过分析瞬时叶绿素荧光诱导动力学曲线,可以获得围绕光系统Ⅱ(photosystemⅡ,PSⅡ)发生的原初光化学反应的信息。通过分析延迟叶绿素荧光诱导动力学和衰减动力学曲线,可以探寻发生电荷重组而产生延迟荧光的不同基团,从而更加直接地了解PSⅡ的状态。而820 nm光反射可以用来检测发生在光系统Ⅰ(photosystemⅠ,PSⅠ)的原初光化学反应。文章简要介绍了这三种动力学的发生原理及其在光合作用研究中的优缺点,并举例说明了瞬时荧光、延迟荧光及820 nm光反射动力学同步测量技术在光合作用研究中的应用,以及三者之间的互补与印证作用。     

快速叶绿素荧光动力学及其在植物抗逆生理研究中的应用

快速叶绿素荧光动力学是研究光合原初反应的无损探针,可监测光合反应的多个事件,并能反应植物的生理状况,可以应用于环境物理及化学条件胁迫对植物的影响(如化肥及除草剂的使用;CO2、O2、O3浓度的变化及温度、光强度胁迫等);也应用于农业生产(如确定耕作方式及日常管理;除草剂、杀虫剂及激素的使用;品种选优等);更是光合生理研究的便捷工具。介绍了快速叶绿素荧光动力学中的JIP-test分析方法,并从光、水分、温度、盐等多个方面介绍了JIP-test方法在植物抗逆生理研究中的应用。     

P515的测量原理、方法和应用

光谱技术已广泛应用于光合研究领域, 如光吸收信号P515和P700氧化还原动力学以及叶绿素荧光等, 可快速、准确地检测植物的光合活性。P515信号广泛存在于高等植物和藻类中, 是类囊体膜上的色素分子吸收光能后, 其吸收光谱发生位移造成。利用光诱导的P515快速和慢速动力学, 可检测PSI和PSII反应中心的比值、ATP合酶的质子传导性、围绕PSI的环式电子传递速率、质子动力势及其组分, 还可通过同步检测叶绿素荧光和P515信号研究光保护机制。该文总结了P515的主要测量原理、方法及其应用, 旨在为深入研究光合作用机理提供技术支持。     

DIGE技术及其在植物蛋白质组学研究中的应用

双向荧光差异凝胶电泳(DIGE)是一项新出现的荧光标记定量蛋白质组学技术,比经典的双向凝胶电泳(2-DE)具有更高的动力学范围和灵敏性。文章综述了DIGE蛋白质组学的基本原理、实验方法、在植物蛋白质组学研究中的应用和局限性,并对DIGE技术的应用做了展望。     

自控闪光动力学光谱仪及其在紫膜光化循环研究中的应用

在对紫膜光化循环过程的研究中,为了了解光化循环过程中间产物的性质,需要测量其瞬态光吸收的变化。动力学光谱仪就是为此目的设计的。根据脉冲光能动力学测量原理和国内外类似仪器的特点,我们研制成用于测量紫膜光循环中具有毫秒级寿命的中间产物变化的闪光动力学光谱仪。本文对该装置和软件作一简单介绍。     

叶绿素荧光分析技术及应用进展

叶绿素荧光动力学技术被称为研究植物光合功能的快速、无损伤探针,已逐渐在环境胁迫对植物光合作用影响研究方面得到应用,随着叶绿素荧光分析技术的进一步发展,其应用领域和研究空间将进一步拓展.本文介绍了叶绿素荧光分析的基本原理,综述了叶绿素荧光分析技术的应用研究进展.     

PMS: Photosystem I electron donor or fluorescence quencher

Light energy harvested by the pigments in Photosystem I (PSI) is used for charge separation in the reaction center (RC), after which the positive charge resides on a special chlorophyll dimer called P700. In studies on the PSI trapping kinetics, P700(+) is usually chemically reduced to re-open the RCs. So far, the information available about the reduction rate and possible chlorophyll fluorescence quenching effects of these reducing agents is limited. This information is indispensible to estimate the fraction of open RCs under known experimental conditions. Moreover, it would be important to understand if these reagents have a chlorophyll fluorescence quenching effects to avoid the introduction of exogenous singlet excitation quenching in the measurements. In this study, we investigated the effect of the commonly used reducing agent phenazine methosulfate (PMS) on the RC and fluorescence emission of higher plant PSI-LHCI. We measured the P700(+) reduction rate for different PMS concentrations, and show that we can give a reliable estimation on the fraction of closed RCs based on these rates. The data show that PMS is quenching chlorophyll fluorescence emission. Finally, we determined that the fluorescence quantum yield of PSI with closed RCs is 4% higher than if the RCs are open.     

Large-scale analysis of chlorophyll fluorescence kinetics in Synechocystis sp. PCC 6803: identification of the factors involved in the modulation of photosystem stoichiometry

Since chlorophyll fluorescence reflects the redox state of photosynthetic electron transport chain, monitoring of chlorophyll fluorescence has been successfully applied for the screening of photosynthesis-related genes. Here we report that the mutants having a defect in the regulation of photosystem stoichiometry could be identified through the simple comparison of the induction kinetics of chlorophyll fluorescence. We made a library containing 500 mutants in the cyanobacterium Synechocystis sp. PCC 6803 with transposon-mediated gene disruption, and the mutants were used for the measurement of chlorophyll fluorescence kinetics for 45 s. We picked up two genes, pmgA and sll1961, which are involved in the modulation of photosystem stoichiometry. The disruptants of the two genes share common characteristics in their fluorescence kinetics, and we searched for mutants that showed such characteristics. Out of six mutants identified so far, five showed a different photosystem stoichiometry under high-light conditions. Thus, categorization based on the similarity of fluorescence kinetics is an excellent way to identify the function of genes.     

A CCD-OMA device for the measurement of complete chlorophyll fluorescence emission spectra of leaves during the fluorescence induction kinetics

Summary A new device for the measurement of complete laser induced fluorescence emission spectra (maxima near 690 and 735 nm) of leaves during the induction of the chlorophyll fluorescence is described. In this the excitation light (cw He/Ne laser, 632.8 nm) is switched on by a fast electro-mechanical shutter which provides an opening time of 1 ms. The emitted fluorescence is imaged onto the entrance slit of a multichannel spectrograph through a red cut-off filter (> 645 nm). A charge coupled device (CCD) sensor with 2048 elements simultaneously detects the complete chlorophyll fluorescence emission spectrum in the 650–800 nm wavelength range. Scanning is accomplished electronically and the integration time for a complete fluorescence emission spectrum can be selected from 10 ms up to 260 ms. Shutter, detector system and data acquisition are controlled by an IBM-PC/AT compatible computer. A maximum of 32 spectra can be measured at selected times during the fluorescence induction kinetics with the shortest time resolution of 10 ms. The instrument permits the determination of various fluorescence parameters:a) the rise-time of the fluorescence to the maximum level fm,b) the changes in the shape of the fluorescence emission spectra during the induction kinetics,c) the induction kinetics in the fluorescence ratio F690/F735 as well asd) the fluorescence decrease ratio Rfd at any wavelength between 650 to 800 nm. These fluorescence parameters provide information about the functioning of photosynthesis. The ratio F690/F735 allows the non-destructive determination of the chlorophyll content of leaves. The application of this instrument in ecophysiological research and stress physiology of plants is outlined.     

Extended depth-of-focus imaging of chlorophyll fluorescence from intact leaves

Imaging dynamic changes in chlorophyll a fluorescence provides a valuable means with which to examine localised changes in photosynthetic function. Microscope-based systems provide excellent spatial resolution which allows the response of individual cells to be measured. However, such systems have a restricted depth of focus and, as leaves are inherently uneven, only a small proportion of each image at any given focal plane is in focus. In this report we describe the development of algorithms, specifically adapted for imaging chlorophyll fluorescence and photosynthetic function in living plant cells, which allow extended-focus images to be reconstructed from images taken in different focal planes. We describe how these procedures can be used to reconstruct images of chlorophyll fluorescence and calculated photosynthetic parameters, as well as producing a map of leaf topology. The robustness of this procedure is demonstrated using leaves from a number of different plant species. This revised version was published online in June 2006 with corrections to the Cover Date.     

An LED-based fluorometer for chlorophyll quantification in the laboratory and in the field

The chlorophyll content is an important experimental parameter in agronomy and plant biology research. In this report, we explore the feasibility of determining total concentration of extracts containing chlorophyll a and chlorophyll b by chlorophyll fluorescence. We found that an excitation at 457?nm results in the same integrated fluorescence emission for a molecule of chlorophyll a and a molecule of chlorophyll b. The fluorescence yield induced by 457?nm is therefore proportional to total molar chlorophyll concentration. Based on this observation, we designed an instrument to determine total chlorophyll concentrations. A single light emitting diode (LED) is used to excite chlorophyll extracts. After passing through a long-pass filter, the fluorescence emission is assessed by a photodiode. We demonstrate that this instrument facilitates the determination of total chlorophyll concentrations. We further extended the functionality of the instrument by including LEDs emitting at 435 and 470?nm wavelengths, thereby preferentially exciting chlorophyll a and chlorophyll b. This instrument can be used to determine chlorophyll a and chlorophyll b concentrations in a variety of organisms containing different ratios of chlorophylls. Monte-Carlo simulations are in agreement with experimental data such that a precise determination of chlorophyll concentrations in carotenoid-containing biological samples containing a concentration of less than 5?nmol/mL total chlorophyll can be achieved.     

Frequently asked questions about in vivo chlorophyll fluorescence: practical issues

The aim of this educational review is to provide practical information on the hardware, methodology, and the hands on application of chlorophyll (Chl) a fluorescence technology. We present the paper in a question and answer format like frequently asked questions. Although nearly all information on the application of Chl a fluorescence can be found in the literature, it is not always easily accessible. This paper is primarily aimed at scientists who have some experience with the application of Chl a fluorescence but are still in the process of discovering what it all means and how it can be used. Topics discussed are (among other things) the kind of information that can be obtained using different fluorescence techniques, the interpretation of Chl a fluorescence signals, specific applications of these techniques, and practical advice on different subjects, such as on the length of dark adaptation before measurement of the Chl a fluorescence transient. The paper also provides the physiological background for some of the applied procedures. It also serves as a source of reference for experienced scientists.     

Excitation kinetics of chlorophyll fluorescence during light-induced greening and establishment of photosynthetic activity of barley seedlings

Excitation kinetics based on feedback regulation of chlorophyll (Chl) fluorescence of leaves measured with the chlorophyll fluorometer, FluoroMeter Modul (FMM), are presented. These kinetics showed the variation of excitation light (laser power, LP) regulated by the feedback mechanism of the FMM, an intelligent Chl fluorometer with embedded computer, which maintains the fluorescence response constant during the 300-s transient between the dark- and lightadapted state of photosynthesis. The excitation kinetics exhibited a rise of LP with different time constants and fluctuations leading to a type of steady state. The variation of excitation kinetics were demonstrated using the example of primary leaves of etiolated barley seedlings (Hordeum vulgare L. cv. Barke) during 48 h of greening in the light with gradual accumulation of Chl and development of photosynthetic activity. The excitation kinetics showed a fast rise followed by a short plateau at ca. 30 s and finally a slow constant increase up to 300 s. Only in the case of 2 h of greening in the light, the curve reached a stable steady state after 75 s followed by a slight decline. The final LP value (at 300 s of illumination) increased up to 12 h of greening and decreased with longer greening times. The active feedback mechanism of the FMM adjusted the excitation light during the measurement to the actual photosynthetic capacity of the individual leaf sample. In this way, the illumination with excessive light was avoided. The novel excitation kinetics can be used to characterize health, stress, disease, and/or product quality of plant material.