An apparatus for measuring photosynthetic quantum yields and quanta absorption spectra of intact plants

Abstract An apparatus is described which consists of an assimilation chamber mounted in the centre of a light-integrating Ulbricht sphere, irradiated through two fibreoptic light pipes. This arrangement provides totally diffuse radiation in the sphere. The quantum flux density in die sphere is measured by an integrating quantaspectrometer connected to the sphere by eight fibreoptic light pipes. The quantaspectrometer gives the spectral and the total quantum flux density in the sphere. By measuring the quantum flux density in the sphere before and after the insertion of a plant the number of absorbed quanta per unit time and plant area can be determined. In addition, the spectral distribution of the absorbed quantum flux density is obtained. CO₂-assimilation per unit time and plant area is determined in the same apparatus. The totally diffuse radiation within the sphere minimizes mutual shading between branches and leaves. Hence, well-defined light-responsecurves of photosynthesis can be obtained by plotting the flux density of C0₂-assimilation as a function of the absorbed quantum flux density, and the quantum yields of intact plants can be calculated. Examples of photosynthetic quantum yield determinations and quanta absorption spectra are given for some plant species.     

Measurement of leaf and canopy photosynthetic CO2 exchange in the field

The principles and limitations of leaf gas exchange measurementsin portable gas exchange systems are described. Attention isgiven to the design and developments in infrared gas analysersused in portable systems, and the basic structure of singleand dual beam instruments is presented. The significance offlow measurement in these systems and the principles of thermalmass flow measurement are illustrated. Considerations of leafarea measurement, chamber design and choice of materials areoutlined. Two specific developments in field gas exchange systemsare described and their significance in field measurements isillustrated with examples. (1) An integrating sphere leaf chamberfor the determination of the quantum yield of photosynthesis,on the basis of absorbed light, is explained and equations forits use are developed. The significance of this approach isillustrated by a comparison of data for contrasting leaves plottedon an absorbed and incident light basis. This measurement oflight-limited photosynthesis is also critical in understandingthe contribution of shaded leaves to canopy photosynthesis.(2) A system for the measurement of canopy photosynthesis fromarable crops and low stature natural vegetation is described.Results from a season-long study of wheat CO₂ exchange are shownto illustrate its application. Key words: Leaf gas exchange, photosynthetic quantum efficiency, infrared gas analysis, canopy photosynthesis, integrating sphere     

Measurement of chlorophyll fluorescence within leaves using a modified PAM Fluorometer with a fiber-optic microprobe

By using a fiber-optic microprobe in combination with a modified PAM Fluorometer, chlorophyll fluorescence yield was measured within leaves with spatial resolution of approximately 20 m. The new system employs a miniature photomultiplier for detection of the pulse-modulated fluorescence signal received by the 20 m fiber tip. The obtained signal/noise ratio qualifies for recordings of fluorescence induction kinetics (Kautsky effect), fluorescence quenching by the saturation pulse method and determination of quantum yield of energy conversion at Photosystem II at different sites within a leaf. Examples of the system performance and of practical applications are given. It is demonstrated that the fluorescence rise kinetics are distinctly faster when chloroplasts within the spongy mesophyll are illuminated as compared to palisade chloroplasts. Photoinhibition is shown to affect primarily the quantum yield of the palisade chloroplasts when excessive illumination is applied from the adaxial leaf side. The new system is envisaged to be used in combination with light measurements within leaves for an assessment of the specific contributions of different leaf regions to overall photosynthetic activity and for an integrative modelling of leaf photosynthesis.This paper is dedicated to Ulrich Heber on the occasion of his 65th birthday, with great respect for his outstanding achievements in photosynthesis research.     

Lateral diffusion of CO2 from shaded to illuminated leaf parts affects photosynthesis inside homobaric leaves

Gas exchange is generally regarded to occur between the leaf interior and ambient air, i.e. in vertical (anticlinal) directions of leaf blades. However, inside homobaric leaves, gas movement occurs also in lateral directions. The aim of the present study was to ascertain whether lateral CO2 diffusion affects leaf photosynthesis when illuminated leaves are partially shaded. Measurements using gas exchange and chlorophyll fluorescence imaging techniques were performed on homobaric leaves of Vicia faba and Nicotiana tabacum or on heterobaric leaves of Glycine max and Phaseolus vulgaris. For homobaric leaves, gas exchange inside a clamp-on leaf chamber was affected by shading the leaf outside the chamber. The quantum yield of photosystem II (Phi(PSII)) was highest directly adjacent to a light/shade border (LSB). Phi(PSII) decreased in the illuminated leaf parts with distance from the LSB, while the opposite was observed for nonphotochemical quenching. These effects became most pronounced at low stomatal conductance. They were not observed in heterobaric leaves. The results suggest that plants with homobaric leaves can benefit from lateral CO2 flux, in particular when stomata are closed (e.g. under drought stress). This may enhance photosynthetic, instead of nonphotochemical, processes near LSBs in such leaves and reduce the photoinhibitory effects of excess light.     

An integrated portable apparatus for the simultaneous field measurement of photosynthetic CO2 and water vapour exchange, light absorption and chlorophyll fluorescence emission of attached leaves

Abstract. A portable apparatus has been constructed to measure simultaneously the quantum yield of CO₂ assimilation, light absorption, chlorophyll fluorescence emission and water vapour exchange of attached intact leaves in the field. The core of the instrument is a light-integrating spherical leaf chamber which includes ports for a light source, photosynthetically active radiation sensor, fluorescence probes and gas inlet and outlet manifolds. Measurement of the quantum flux inside the empty chamber and with a leaf present allows determination of leaf absorptance. An open gas exchange system is employed using an infra-red analyser to measure leaf CO₂ exchange. Using a DC white light source the quantum yield of CO₂ assimilation based on absorbed light (φ_abs) may be determined rapidly in either ambient air or artificial gas mixtures. Inclusion of capacitance humidity probes into the gas inlet and outlet ports allows simultaneous determination of water vapour exchange and subsequent estimation of stomatal conductance to CO₂ and intercellular CO₂ concentration. Measurement of fluorescence emission by the sample leaf exposed to white light is achieved by a modulated fluorescence detection system. In addition to determination of the minimal, maximal and variable fluorescence levels, a further analysis allows the photochemical and non-photochemical components of fluorescence quenching, to be estimated. The theory and design of this apparatus is described in detail. The use of the apparatus in the field is demonstrated through a study of the photosynthetic performance of a maize and bean crop during the growing season and by analysis of the photosynthetic performance of crops subjected to nitrogen-stress and a herbicide treatment.     

Absolute quantum yield measurement of powder samples

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and research of illumination, AV equipment, organic EL material, films, filters and fluorescent probes for bio-industry. Quantum yield is calculated as the ratio of the number of photons absorbed, to the number of photons emitted by a material. The higher the quantum yield, the better the efficiency of the fluorescent material. For the measurements featured in this video, we will use the Hitachi F-7000 fluorescence spectrophotometer equipped with the Quantum Yield measuring accessory and Report Generator program. All the information provided applies to this system. Measurement of quantum yield in powder samples is performed following these steps: 1. Generation of instrument correction factors for the excitation and emission monochromators. This is an important requirement for the correct measurement of quantum yield. It has been performed in advance for the full measurement range of the instrument and will not be shown in this video due to time limitations. 2. Measurement of integrating sphere correction factors. The purpose of this step is to take into consideration reflectivity characteristics of the integrating sphere used for the measurements. 3. Reference and Sample measurement using direct excitation and indirect excitation. 4. Quantum Yield calculation using Direct and Indirect excitation. Direct excitation is when the sample is facing directly the excitation beam, which would be the normal measurement setup. However, because we use an integrating sphere, a portion of the emitted photons resulting from the sample fluorescence are reflected by the integrating sphere and will re-excite the sample, so we need to take into consideration indirect excitation. This is accomplished by measuring the sample placed in the port facing the emission monochromator, calculating indirect quantum yield and correcting the direct quantum yield calculation. 5. Corrected quantum yield calculation. 6. Chromaticity coordinates calculation using Report Generator program. The Hitachi F-7000 Quantum Yield Measurement System offer advantages for this application, as follows: High sensitivity (S/N ratio 800 or better RMS). Signal is the Raman band of water measured under the following conditions: Ex wavelength 350 nm, band pass Ex and Em 5 nm, response 2 sec), noise is measured at the maximum of the Raman peak. High sensitivity allows measurement of samples even with low quantum yield. Using this system we have measured quantum yields as low as 0.1 for a sample of salicylic acid and as high as 0.8 for a sample of magnesium tungstate. Highly accurate measurement with a dynamic range of 6 orders of magnitude allows for measurements of both sharp scattering peaks with high intensity, as well as broad fluorescence peaks of low intensity under the same conditions. High measuring throughput and reduced light exposure to the sample, due to a high scanning speed of up to 60,000 nm/minute and automatic shutter function. Measurement of quantum yield over a wide wavelength range from 240 to 800 nm. Accurate quantum yield measurements are the result of collecting instrument spectral response and integrating sphere correction factors before measuring the sample. Large selection of calculated parameters provided by dedicated and easy to use software. During this video we will measure sodium salicylate in powder form which is known to have a quantum yield value of 0.4 to 0.5.     

Neurotransmitter Specific,Cellular-Resolution Functional Brain Mapping Using Receptor Coated Nanoparticles: Assessment of the Possibility

Receptor coated resonant nanoparticles and quantum dots are proposed to provide a cellular-level resolution image of neural activities inside the brain. The functionalized nanoparticles and quantum dots in this approach will selectively bind to different neurotransmitters in the extra-synaptic regions of neurons. This allows us to detect neural activities in real time by monitoring the nanoparticles and quantum dots optically. Gold nanoparticles (GNPs) with two different geometries (sphere and rod) and quantum dots (QDs) with different sizes were studied along with three different neurotransmitters: dopamine, gamma-Aminobutyric acid (GABA), and glycine. The absorption/emission spectra of GNPs and QDs before and after binding of neurotransmitters and their corresponding receptors are reported. The results using QDs and nanorods with diameter 25nm and aspect rations larger than three were promising for the development of the proposed functional brain mapping approach.     

The analysis of the mechanosensory origin of the infrared sensilla in Melanophila acuminata (Coeloptera; Buprestidae) adduces new insight into the transduction mechanism

The thoracic infrared (IR) sensilla of the pyrophilous jewel beetle Melanophila acuminata most likely have evolved from hair mechanoreceptors (sensilla trichodea). To further elucidate the sensory transduction mechanism, the morphology of IR sensilla and of neighbouring hair mechanoreceptors was investigated by using conventional electron microscopical techniques (SEM, TEM) in combination with focused ion beam milling (FIB). It was assumed that any deviation from the bauplan of a sensillum trichodeum is of particular concern for the transduction of IR radiation into a mechanical stimulus. Thus, the structures supposed to be relevant for stimulus uptake and transduction were homologized. Compared to a hair mechanoreceptor, an IR sensillum shows the following special features: (i) the formation of a complex cuticular sphere instead of the bristle; the sphere consists of an outer exocuticular shell as well as of an inner porous mesocuticular part. (ii) The enclosure of the dendritic tip of the mechanosensitive neuron inside the sphere in a fluid-filled inner pressure chamber which is connected with a system of microcavities and nanocanals in the mesocuticular part. Hence we propose that an IR sensillum most probably acts as a microfluidic converter of infrared radiation into an increase in internal pressure inside the sphere which is measured by the mechanosensitive neuron.     

Excitation kinetics during induction of chlorophyll <Emphasis Type="Italic">a</Emphasis> fluorescence

We present a chlorophyll fluorometer module system which adapts the intensity to the individual leaf sample by adjusting the quantum flux density of the excitation light so that the fluorescence signal is kept constant. This is achieved by means of a feedback power adjustment of the fluorescence exciting laser diode. Thus, the intensity of the excitation light is adapted to the actual need of a particular sample for quantum conversion without applying exaggeratedly high quantum flux density. We demonstrate the influence of the initial laser power chosen at the onset of irradiation and kept constant during fluorescence rise transient within the first second. Examples are shown for measuring upper and lower leaf sides, a single leaf with different pre-darkening periods, as well as yellow, light green and dark green leaves. The novel excitation kinetics during the induction of chlorophyll fluorescence can be used to study the yield and regulation of photosynthesis and its related non-photochemical processes for an individual leaf. It allows not only to sense the present state of pre-darkening or pre-irradiation but also the light environment the leaf has experienced during its growth and development. Thus, the individual physiological capacity and plasticity of each leaf sample can be sensed being of high importance for basic and applied ecophysiological research which makes this new methodology both innovative and informative.     

The relationship between photosystem II efficiency and quantum yield for CO(2) assimilation is not affected by nitrogen content in apple leaves

Bench-grafted Fuji/M.26 apple (Malus domestica Borkh.) trees were fertigated with different concentrations of nitrogen by using a modified Hoagland's solution for 45 d. CO(2) assimilation and photosystem II (PSII) quantum efficiency in response to incident photon flux density (PFD) were measured simultaneously in recent fully expanded leaves under low O(2) (2%) and saturated CO(2) (1300 micromol mol(-1)) conditions. A single curvilinear relationship was found between true quantum yield for CO(2) assimilation and PSII quantum efficiency for leaves with a wide range of leaf N content. The relationship was linear up to a quantum yield of approximately 0.05 mol CO(2) mol(-1) quanta. It then became curvilinear with a further rise in quantum yield in response to decreasing PFD. This relationship was subsequently used as a calibration curve to assess the rate of non-cyclic electron transport associated with Rubisco and the partitioning of electron flow between CO(2) assimilation and photorespiration in different N leaves in response to intercellular CO(2) concentration (C(i)) under normal O(2) conditions. Both the rate of non-cyclic electron flow and the rate of electron flow to CO(2) or O(2) increased with increasing leaf N at any given C(i). The percentage of non-cyclic electron flow to CO(2) assimilation, however, remained the same regardless of leaf N content. As C(i) increased, the percentage of non-cyclic electron flow to CO(2) assimilation increased. In conclusion, the relationship between PSII quantum efficiency and quantum yield for CO(2) assimilation and the partitioning of electron flow between CO(2) assimilation and photorespiration are not affected by N content in apple leaves.     

Photosynthesis in intact leaves of C3 plants: Physics,physiology and rate limitations

The instantaneous rate of photosynthetic CO₂ assimilation in C₃ plants has generally been studied in model systems such as isolated chloroplasts and algae. From these studies and from theoretical analyses of gas exchange behavior it is now possible to study the biochemistry of photosynthesis in intact leaves using a combination of methods, most of which are nondestructive. The limitations to the rate of photosynthesis can be divided among three general classes: (1) the supply or utilization of CO₂, (2) the supply or utilization of light, and (3) the supply or utilization of phosphate. The first limitation is most readily studied by determining how the CO₂ assimilation rate varies with the partial pressure of CO₂ inside the leaf. The second limitation can be studied by determining the quantum requirement of photosynthesis. The third limitation is most easily detected as a loss of O₂ sensitivity of photosynthesis. Measurement of fluorescence from intact leaves can give additional information about the various limitations. These methods are all non-destructive and so can be observed repeatedly as the environment of a leaf is changed. In addition, leaves can be quick-frozen and metabolite concentrations then measured to give more information about the limitations to intact leaf photosynthesis rates. In this review the physics and biochemistry of photosynthesis in intact C₃ leaves, and the interface between physiology and photosynthesis—triose phosphate utilization—are discussed.     

A computer model of temperature distribution inside a lossy sphere after microwave radiation

The temperature distribution inside a lossy sphere resulting from the absorption of microwave energy was approximated by successive numerical iterations, of the thermal energy equation. Heat transfer within the sphere by conduction was considered. In the model energy was not dissipated by convection but was contained in the sphere for over 200 seconds. Exposure of a 5-cm sphere to 3,000 MHz at 30 mW/cm² for 200 seconds was calculated to produce a temperature rise of 0.56°C near the front surface.     

Self-organization in and on biological spheres

Three biological settings involving self-organization performed by the Turing-Child field inside a sphere and on its surface are considered. In the first setting the interior of a sphere made up of cells communicating via gap junctions is considered. It is suggested that the Turing-Child self-organization is the cause of radial polarization, the first differentiation of an early mammalian embryo. In the second setting, the Turing example of gastrulation of a hollow cellular sphere is considered. It is shown that Child's experimental patterns are predicted and explained by the Turing-Child theory. The third setting is the interior of a biological cell, and it is suggested that it is the self-organization of the Turing-Child field that causes the formation of the mitotic spindle.     

Evolutionarily stable leaf area production in plant populations

Using an analytical model, it was shown that for a given amount of nitrogen in the canopy of a stand (N(T)), there exists an evolutionarily stable leaf area index (ES-LAI), and therefore an evolutionarily stable average leaf nitrogen content (n(ES)(av);n(ES)(av) =N(T)/ES-LAI), at which no individual plant in the stand can increase its photosynthesis by changing its leaf area. It was also shown that this ES-LAI is always greater than the optimal LAI that maximizes photosynthesis per unit N(T) of the stand. This illustrates that the canopy structure that maximizes photosynthesis of a population is not the same as the canopy structure that maximizes photosynthesis of individuals within a population. It was further derived that the ES-LAI at given N(T) increases with the ratio between the light-saturated photosynthesis and the N content per unit leaf area (leaf-PPNUE) and that it decreases with the canopy extinction coefficient for light (K(L)), the light availability and the apparent quantum yield (phi). These hypotheses were tested by comparing calculated ES-LAI and n(ES)(av) values to actual LAIs and leaf N contents measured for stands of a large variety of herbaceous plants. There was a close correspondence between the calculated and measured values. As predicted by the model, plants with high leaf-PPNUEs produced more leaf area per unit nitrogen than those with low leaf-PPNUEs while plants with horizontal leaves, forming stands with higher K(L) values, produced less leaf area than those with more vertically inclined leaves. These results suggest that maximization of individual plant photosynthesis per unit of nitrogen plays an important role in determining leaf area production of plants and the resulting canopy structure of stands of vegetation. They further suggest this optimization to be a mechanism by which leaf traits such as leaf-PPNUE and leaf inclination angle are causally related to structural characteristics of the population, i.e. the leaf area index of the stand.     

Self-organization in and on biological spheres

Three biological settings involving self-organization performed by the Turing-Child field inside a sphere and on its surface are considered. In the first setting the interior of a sphere made up of cells communicating via gap junctions is considered. It is suggested that the Turing-Child self-organization is the cause of radial polarization, the first differentiation of an early mammalian embryo. In the second setting, the Turing example of gastrulation of a hollow cellular sphere is considered. It is shown that Child's experimental patterns are predicted and explained by the Turing-Child theory. The third setting is the interior of a biological cell, and it is suggested that it is the self-organization of the Turing-Child field that causes the formation of the mitotic spindle.     

The coordination chemistry of the structural zinc ion in alcohol dehydrogenase studied by ab initio quantum chemical calculations

The coordination chemistry of the structural zinc ion in horse liver alcohol dehydrogenase has been examined by quantum chemical geometry optimisations. It is shown that all four cysteine ligands are deprotonated in the enzyme, not only two of them as has been suggested. The Zn-S bond lengths are very sensitive to the theoretical treatment; in vacuum they are predicted to be 15 pm longer than in the crystal structure. Half of this discrepancy is due to electronic correlation, the rest can be attributed to screening of the negative sulphide charges by the enzyme, in particular by N-H-S hydrogen bonds. The potential surface is rather flat, so the large difference in geometry between the crystal and the vacuum structure corresponds to an energy change of less than 35 kJ/mol. The experimental bond lengths can be reproduced only with methods that account explicitly for the enzyme. A dielectric continuum model gives bond lengths which are too long, indicating that the enzyme solvates the coordination sphere better than water. Thus, the structural zinc ion can be used as a sensitive test of methods which try to model the surrounding medium in quantum chemical computations.     

The shapes of backbones of chain molecules: Three-dimensional characterization by spherical shape maps

In this work we present a new method to characterize the shape of flexible chain molecules. The procedure associates a sphere and a spherical shape map with each given molecular backbone. Each point on the sphere is classified according to the crossing pattern obtained when the backbone is looked at along a direction defined by the center of the sphere and the chosen point. The approach is simple to implement. It consists of the following steps: (a) enclosing the backbone by a sphere, whose center is the geometrical center of the backbone; (b) projecting the backbone onto a plane tangent to the sphere at the given point; and (c) characterizing the resulting planar curve by a vector specifying the number and handedness type of the crossings. When the procedure is repeated for all points on the spherical surface, the latter is divided into regions that are equivalence classes of points, corresponding to directions from where the backbone has the same overcrossing pattern. The computation of these equivalence classes is performed automatically by the computer, by determining the boundary of the regions characterized by different crossing vectors. The characterization provided is thus direction independent since it takes into account all possible directions from where a backbone can be analyzed. The procedure is illustrated for a number of supersecondary protein structures and small proteins. We find that a characterization of substructures can be obtained in terms of the arrangement of the equivalence region for the viewing directions from where the backbone shows no crossings. For instance, an α-helix is represented by a spherical map with a small “band” region of no crossings perpendicular to the helical axis. Other supersecondary structural features are described in a similar fashion. A number of refinements of the method, based on the distances between crossings, are also given for the case of irregular backbones. © 1992 John Wiley & Sons, Inc.     

The effects of UV-irradiation of barley plants on the morphophysiological parameters and productivity of the offsprings

In a greenhouse experiment effects have been studied the UV-B-irradiation of barley (intensity of biologically active UV-B-radiation was 0.15, 0.29 and 0.45 W/m2) on the morphophysiologic parameters, productivity and reproductive sphere of offsprings of two succeeding generations. It has been shown that along with a decrease in the specific leaf mass and biomass M1-generation plants exhibited an increase in the stem height and assimilative leaf area. The reduction of grain yield in offsprings of the first post-radiation generation was mainly caused by the reduced size in the reproductive organs and by reduced number of caryopsides in a ear. The observed effects were independent of the UV-B exposure levels to the precursors. The aftereffect of UV-B-irradiation for M2-generation plants shown itself as changes in the specific leaf area, less pronounced compared to the precursors, and reduction in the vegetative mass and grain yield, most significant in offsprings of plants exposed to the maximum UV-B levels. Disturbances in the reproductive sphere appeared as reduction in the size of developing grains. The data derived suggest that long-term effects of UV-B-irradiation are possible not only in the first but also in the second generation of impacted plants.     

Biologically enhanced sampling geometric docking and backbone flexibility treatment with multiconformational superposition

An efficient biologically enhanced sampling geometric docking method is presented based on the FTDock algorithm to predict the protein-protein binding modes. The active site data from different sources, such as biochemical and biophysical experiments or theoretical analyses of sequence data, can be incorporated in the rotation-translation scan. When discretizing a protein onto a 3-dimensional (3D) grid, a zero value is given to grid points outside a sphere centered on the geometric center of specified residues. In this way, docking solutions are biased toward modes where the interface region is inside the sphere. We also adopt a multiconformational superposition scheme to represent backbone flexibility in the proteins. When these procedures were applied to the targets of CAPRI, a larger number of hits and smaller ligand root-mean-square deviations (RMSDs) were obtained at the conformational search stage in all cases, and especially Target 19. With Target 18, only 1 near-native structure was retained by the biologically enhanced sampling geometric docking method, but this number increased to 53 and the least ligand RMSD decreased from 8.1 A to 2.9 A after performing multiconformational superposition. These results were obtained after the CAPRI prediction deadlines.