Frequently asked questions about chlorophyll fluorescence,the sequel

Using chlorophyll (Chl) a fluorescence many aspects of the photosynthetic apparatus can be studied, both in vitro and, noninvasively, in vivo. Complementary techniques can help to interpret changes in the Chl a fluorescence kinetics. Kalaji et al. (Photosynth Res 122:121–158, 2014a) addressed several questions about instruments, methods and applications based on Chl a fluorescence. Here, additional Chl a fluorescence-related topics are discussed again in a question and answer format. Examples are the effect of connectivity on photochemical quenching, the correction of F _V /F _M values for PSI fluorescence, the energy partitioning concept, the interpretation of the complementary area, probing the donor side of PSII, the assignment of bands of 77 K fluorescence emission spectra to fluorescence emitters, the relationship between prompt and delayed fluorescence, potential problems when sampling tree canopies, the use of fluorescence parameters in QTL studies, the use of Chl a fluorescence in biosensor applications and the application of neural network approaches for the analysis of fluorescence measurements. The answers draw on knowledge from different Chl a fluorescence analysis domains, yielding in several cases new insights.     

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.     

Frequently asked questions about the public health service policy on humane care and use of laboratory animals

The authors answer eight questions commonly asked of the Office of Laboratory Animal Welfare concerning Public Health Service Policy on Humane Care of Use of Laboratory Animals.     

Matching loci surveyed to questions asked in phylogeography

Although mitochondrial DNA (mtDNA) has long been used for assessing genetic variation within and between populations, its workhorse role in phylogeography has been criticized owing to its single-locus nature. The only choice for testing mtDNA results is to survey nuclear loci, which brings into contrast the difference in locus effective size and coalescence times. Thus, it remains unclear how erroneous mtDNA-based estimates of species history might be, especially for evolutionary events in the recent past. To test the robustness of mtDNA and nuclear sequences in phylogeography, we provide one of the largest paired comparisons of summary statistics and demographic parameters estimated from mitochondrial, five Z-linked and 10 autosomal genes of 30 avian species co-distributed in the Caucasus and Europe. The results suggest that mtDNA is robust in estimating inter-population divergence but not in intra-population diversity, which is sensitive to population size change. Here, we provide empirical evidence showing that mtDNA was more likely to detect population divergence than any other single locus owing to its smaller N_e and thus faster coalescent time. Therefore, at least in birds, numerous studies that have based their inferences of phylogeographic patterns solely on mtDNA should not be readily dismissed.     

Advanced fluorescence technologies help to resolve long-standing questions about microbial vitality

Advances in fundamental physical and optical principles applied to novel fluorescence methods are currently resulting in rapid progress in cell biology and physiology. Instrumentation devised in pioneering laboratories is becoming commercially available, and study findings are now becoming accessible. The first results have concerned mainly higher eukaryotic cells but many more developments can be expected, especially in microbiology. Until now, some important problems of cell physiology have been difficult to investigate due to interactions between probes and cells, excretion of probes from cells and the inability to make in situ observations deep within the cell, within tissues and structures. These technologies will enable microbiologists to address these topics. This Review aims at introducing the limits of current physiology evaluation techniques, the principles of new fluorescence technologies and examples of their use in this field of research for evaluating the physiological state of cells in model media, biofilms or tissue environments. Perspectives on new imaging technologies, such as super-resolution imaging and non-linear highly sensitive Raman microscopy, are also discussed. This review also serves as a reference to those wishing to explore how fluorescence technologies can be used to understand basic cell physiology in microbial systems.     

Modeling chlorophyll a fluorescence transient: Relation to photosynthesis

To honor Academician Alexander Abramovitch Krasnovsky, we present here an educational review on the relation of chlorophyll a fluorescence transient to various processes in photosynthesis. The initial event in oxygenic photosynthesis is light absorption by chlorophylls (Chls), carotenoids, and, in some cases, phycobilins; these pigments form the antenna. Most of the energy is transferred to reaction centers where it is used for charge separation. The small part of energy that is not used in photochemistry is dissipated as heat or re-emitted as fluorescence. When a photosynthetic sample is transferred from dark to light, Chl a fluorescence (ChlF) intensity shows characteristic changes in time called fluorescence transient, the OJIPSMT transient, where O (the origin) is for the first measured minimum fluorescence level; J and I for intermediate inflections; P for peak; S for semi-steady state level; M for maximum; and T for terminal steady state level. This transient is a real signature of photosynthesis, since diverse events can be related to it, such as: changes in redox states of components of the linear electron transport flow, involvement of alternative electron routes, the build-up of a transmembrane pH gradient and membrane potential, activation of different nonphotochemical quenching processes, activation of the Calvin-Benson cycle, and other processes. In this review, we present our views on how different segments of the OJIPSMT transient are influenced by various photosynthetic processes, and discuss a number of studies involving mathematical modeling and simulation of the ChlF transient. A special emphasis is given to the slower PSMT phase, for which many studies have been recently published, but they are less known than on the faster OJIP phase.     

Biological nitrogen fixation (BNF): Commonly asked questions and answers

J.P. Roskoski was formerly with the NifTAL Project, Department of Agronomy and Soil Science, College of Tropical Agriculture and Human Resources, University of Hawaii, Hawaii, USA     

A perspective on cold enzymes: current knowledge and frequently asked questions.

Studies on psychrophilic enzymes to determine the structural features important for cold-activity have attracted increased attention in the last few years. This enhanced interest is due to the attractive properties of such proteins, i.e. a high specific activity and a low thermal stability, and thus, these enzymes constitute a tremendous potential for fundamental research and biotechnological applications. This review examines the impact of low temperatures on life, the diversity of adaptation to counteract these effects and gives an overview of the features proposed to account for low thermal stability and cold-activity, following the chronological order of the catalytic cycle phases. Moreover, we present an overview of recent techniques used in the analysis of the flexibility of a protein structure which is an important concept in cold-adaptation; an overview of biotechnological potential of psychrophilic enzymes and finally, a few frequently asked questions about cold-adaptation and their possible answers.     

Paramagnetic fluorescence quenching in chlorophyll a containing vesicles: Evidence for the localization of chlorophyll

Spinprobes (fatty acids, androstane, cholestane) that differ in the location of the paramagnetic centre relative to the polar membrane surface have been incorporated into lipid vesicles containing chlorophyll a. Quenching of the Chl a fluorescence is observed following the Stern-Vollmer-relationship. Quenching is more effective with spinprobes carrying the nitroxide group near the polar head groups of the lipid moiety. Quenching is also observed using a water soluble spinlabel. The porphyrin ring of the Chl a molecules is suggested to be localized in the polar head group region of the bilayer membrane.     

Estimating the contribution of Photosystem I to total leaf chlorophyll fluorescence

Chlorophyll a fluorescence characteristics were investigated in 12 species and 2 hybrids from the genus Flaveria exhibiting C₃, C₃–C₄ intermediate, or C₄ photosynthesis, and in the C₄ species Zea mays. At room temperature, the variable fluorescence divided by the maximum fluorescence (F_V/F_M) of dark-adapted leaves decreased from C₃ to C₄ plants. This trend was qualitatively paralleled by an increase of the 735 nm peak relative to the 685 nm peak (F735/F685) of fluorescence emission spectra measured at low temperature (77 K). The variations were analysed using a quantitative model that takes into account higher PS I fluorescence in C₄ plants than in C₃ plants. The model predicts a linear correlation between 1/(F_V/F_M) and F735/F685, and was experimentally confirmed. From linear regression analysis, the F_V/F_M of PS II was calculated to be 0.88. By comparing the F_V/F_M of PS II with the F_V/F_M from leaves, the PS I contribution to total F₀ fluorescence at wavelengths greater than 700 nm was determined to be about 30% and 50% in C₃ and C₄ plants, respectively. The corresponding values for the F_M fluorescence were 6% and 12%. It is concluded that the effects of PS I fluorescence are significant and should be taken into account when analysing fluorescence data.     

Using chlorophyll fluorescence to monitor yields of microalgal production

A monitoring system for microalgal production was developed over a 12:12-h light:dark cycle at a steady state of growth to test the feasibility of estimating carbon production using the fluorescence method. An empirical linear relationship between the electron transport rate, based on the fluorescence method, and carbon assimilation, based on the conventional carbon method, was successfully obtained. The results demonstrate the relevance of the electron transport rate in determining carbon production of microalgae under steady-state growth conditions.     

黄土高原植被日光诱导叶绿素荧光对气象干旱的响应

随着气候变化的加剧,干旱的频率、持续时间以及发生范围都越来越严重,探索植被光合对干旱的响应以及气象因子对植被光合的影响对于人们如何应对干旱具有重要意义。基于遥感的日光诱导叶绿素荧光(SIF)具有对干旱条件下区域植被光合作用进行早期监测和准确评估的潜力。本研究基于星载SIF和标准化降水蒸散发指数(SPEI)研究了黄土高原地区2001—2017年生长季内(4—10月)植被光合作用对干旱的响应关系及其受气象因子的影响程度。结果表明: 黄土高原地区植被生长季内SIF与SPEI呈显著正相关关系的区域占比为87.8%,其中,半干旱地区植被光合对干旱的响应较敏感,半湿润地区敏感性较低。不同类型植被光合对干旱的响应存在差异,草地对干旱响应的敏感性最高,响应最强的SPEI时间尺度为3～4个月;林地的敏感性最低,SPEI时间尺度为3～10个月。气象因子与SIF存在显著的相关关系,其中,温度和降雨是影响黄土高原植被光合的重要影响因子,光合有效辐射的影响模式与温度相似。黄土高原地区生长季内不同的气候和植被类型条件下,植被光合所受干旱及各气象因素的影响存在较大差异。     

Screening for depression in primary care with two verbally asked questions: cross sectional study

Objective To determine the diagnostic accuracy of two verbally asked questions for screening for depression.Design Cross sectional criterion standard validation study.Setting 15 general practices in New Zealand.Participants 421 consecutive patients not taking psychotropic drugs.Main outcome measures Sensitivity, specificity, and likelihood ratios of the two questions compared with the computerised composite international diagnostic interview.Results The two screening questions showed a sensitivity and specificity of 97% (95% confidence interval, 83% to 99%) and 67% (62% to 72%), respectively. The likelihood ratio for a positive test was 2.9 (2.5 to 3.4) and the likelihood ratio for a negative test was 0.05 (0.01 to 0.35). Overall, 37% (157/421) of the patients screened positive for depression.Conclusion Two verbally asked questions for screening for depression would detect most cases of depression in general practice. The questions have the advantage of brevity. As treatment is more likely when doctors make the diagnosis, these questions may have even greater utility.     

Relaxation of the non-photochemical chlorophyll fluorescence quenching in diatoms: kinetics,components and mechanisms

Diatoms are especially important microorganisms because they constitute the larger group of microalgae. To survive the constant variations of the light environment, diatoms have developed mechanisms aiming at the dissipation of excess energy, such as the xanthophyll cycle and the non-photochemical chlorophyll (Chl) fluorescence quenching. This contribution is dedicated to the relaxation of the latter process when the adverse conditions cease. An original nonlinear regression analysis of the relaxation of non-photochemical Chl fluorescence quenching, qN, in diatoms is presented. It was used to obtain experimental evidence for the existence of three time-resolved components in the diatom Phaeodactylum tricornutum: qNf, qNi and qNs. qNf (s time-scale) and qNs (h time-scale) are exponential in shape. By contrast, qNi (min time-scale) is of sigmoidal nature and is dominant among the three components. The application of metabolic inhibitors (dithiothreitol, ammonium chloride, cadmium and diphenyleneiodonium chloride) allowed the identification of the mechanisms on which each component mostly relies. qNi is linked to the relaxation of the ΔpH gradient and the reversal of the xanthophyll cycle. qNs quantifies the stage of photoinhibition caused by the high light exposure, qNf seems to reflect fast conformational changes within thylakoid membranes in the vicinity of the photosystem II complexes.     

关于氧气与呼吸作用几个问题的解答

从生物能学角度、生物化学氧化还原电势角度及化学反应平衡角度,解释O2为什么会抑制无氧呼吸;同时对氧气为什么会“杀死”专性厌氧生物;厌氧生物对氧气的耐受性为何不一样做出了阐述。