A possibility for non-invasive diagnosis of superficial scald in 'Rocha' pear based on chlorophyll a fluorescence, colorimetry, and the relation between alpha-farnesene and conjugated trienols

This study aimed to identify physiological markers in superficially scalded 'Rocha' pear (Pyrus communis L 'Rocha') that would relate to chlorophyll a fluorescence (CF), allowing a non-invasive diagnosis of the disorder. Conditions chosen before shelf life provided two fruit groups with different developing patterns and severity of superficial scald: T fruit fully developed the disorder in storage, while C fruit developed it progressively throughout shelf life. Principal component analysis (PCA) of all the measured variables, and simple linear correlations among several major parameters and scald index (SI)/shelf life showed that scald and ripening/aging were concurring processes, and that it was not possible to isolate a particular variable that could deliver a direct non-invasive diagnosis of the disorder. For both fruit groups the SI resulted from the balance between the reducing power (OD200) and the content of conjugated trienols (CTos) and alpha-farnesene (alpha-Farn) in the fruit peel. At OD200 > 150 there was a linear relationship between CTos and OD200, suggesting that the level of antioxidants was self-adjusted in order to compensate the CTos level. However, at OD200 < 150 this relationship disappeared. A consistent linear relationship between dos and alpha-Farn existed throughout shelf life in both fruit groups, contrarily to the early storage stage, when those compounds do not relate linearly. The CF variables F-0, F-v/F-m, and the colorimetric variables, L* and h degrees were used in multi-linear regressions with other physiological variables. The regressions were made on one of the fruit groups and validated through the other. Reliable regressions to alpha-Farn and CTos were obtained (R approximate to 0.6; rmsec approximate to rmsep). Our results suggest that a model based on CF and colorimetric parameters could be used to diagnose non-invasively both the contents and the relationship between alpha-Farn and CTos and hence the stage of scald development. (C) 2011 Elsevier By. All rights reserved.

A possibility for non-invasive diagnosis of superficial scald in 'Rocha' pear based on chlorophyll a fluorescence, colorimetry, and the relation between alpha-farnesene and conjugated trienols

This study aimed to identify physiological markers in superficially scalded 'Rocha' pear (Pyrus communis L 'Rocha') that would relate to chlorophyll a fluorescence (CF), allowing a non-invasive diagnosis of the disorder. Conditions chosen before shelf life provided two fruit groups with different developing patterns and severity of superficial scald: T fruit fully developed the disorder in storage, while C fruit developed it progressively throughout shelf life. Principal component analysis (PCA) of all the measured variables, and simple linear correlations among several major parameters and scald index (SI)/shelf life showed that scald and ripening/aging were concurring processes, and that it was not possible to isolate a particular variable that could deliver a direct non-invasive diagnosis of the disorder. For both fruit groups the SI resulted from the balance between the reducing power (OD200) and the content of conjugated trienols (CTos) and alpha-farnesene (alpha-Farn) in the fruit peel. At OD200 > 150 there was a linear relationship between CTos and OD200, suggesting that the level of antioxidants was self-adjusted in order to compensate the CTos level. However, at OD200 < 150 this relationship disappeared. A consistent linear relationship between dos and alpha-Farn existed throughout shelf life in both fruit groups, contrarily to the early storage stage, when those compounds do not relate linearly. The CF variables F-0, F-v/F-m, and the colorimetric variables, L* and h degrees were used in multi-linear regressions with other physiological variables. The regressions were made on one of the fruit groups and validated through the other. Reliable regressions to alpha-Farn and CTos were obtained (R approximate to 0.6; rmsec approximate to rmsep). Our results suggest that a model based on CF and colorimetric parameters could be used to diagnose non-invasively both the contents and the relationship between alpha-Farn and CTos and hence the stage of scald development. (C) 2011 Elsevier By. All rights reserved.

Fluorescence properties of Baltic Sea phytoplankton

To obtain data on phytoplankton dynamics with improved spatial and temporal resolution, and at reduced cost, traditional phytoplankton monitoring methods have been supplemented with optical approaches. In this thesis, I have explored various fluorescence-based techniques for detection of phytoplankton abundance, taxonomy and physiology in the Baltic Sea. In algal cultures used in this thesis, the availability of nitrogen and light conditions caused changes in pigmentation, and consequently in light absorption and fluorescence properties of cells. In the Baltic Sea, physical environmental factors (e.g. mixing depth, irradiance and temperature) and related seasonal succession in the phytoplankton community explained a large part of the seasonal variability in the magnitude and shape of Chlorophyll a (Chla)-specific absorption. The variability in Chla-specific fluorescence was related to the abundance of cyanobacteria, the size structure of the phytoplankton community, and absorption characteristics of phytoplankton. Cyanobacteria show very low Chla-specific fluorescence. In the presence of eukaryotic species, Chla fluorescence describes poorly cyanobacteria. During cyanobacterial bloom in the Baltic Sea, phycocyanin fluorescence explained large part of the variability in Chla concentrations. Thus, both Chla and phycocyanin fluorescence were required to predict Chla concentration. Phycobilins are major light harvesting pigments for cyanobacteria. In the open Baltic Sea, small picoplanktonic cyanobacteria were the main source of phycoerythrin fluorescence and absorption signal. Large filamentous cyanobacteria, forming harmful blooms, were the main source of the phycocyanin fluorescence signal and typically their biomass and phycocyanin fluorescence were linearly related. Using phycocyanin fluorescence, dynamics of cyanobacterial blooms can be detected at high spatial and seasonal resolution not possible with other methods. Various taxonomic phytoplankton pigment groups can be separated by spectral fluorescence. I compared multivariate calibration methods for the retrieval of phytoplankton biomass in different taxonomic groups. Partial least squares regression method gave the closest predictions for all taxonomic groups, and the accuracy was adequate for phytoplankton bloom detection. Variable fluorescence has been proposed as a tool to study the physiological state of phytoplankton. My results from the Baltic Sea emphasize that variable fluorescence alone cannot be used to detect nutrient limitation of phytoplankton. However, when combined with experiments with active nutrient manipulation, and other nutrient limitation indices, variable fluorescence provided valuable information on the physiological responses of the phytoplankton community. This thesis found a severe limitation of a commercial fast repetition rate fluorometer, which couldn t detect the variable fluorescence of phycoerythrin-lacking cyanobacteria. For these species, the Photosystem II absorption of blue light is very low, and fluorometer excitation light did not saturate Photosystem II during a measurement. This thesis encourages the use of various in vivo fluorescence methods for the detection of bulk phytoplankton biomass, biomass of cyanobacteria, chemotaxonomy of phytoplankton community, and phytoplankton physiology. Fluorescence methods can support traditional phytoplankton monitoring by providing continuous measurements of phytoplankton, and thereby strengthen the understanding of the links between biological, chemical and physical processes in aquatic ecosystems.

Research on the ultrafast fluorescence property of thylakoid membranes of the wild-type and mutant rice

A high yielding rice variety mutant (Oryza sativa L., Zhenhui 249) with low chlorophyll b (Chl b) has been discovered in natural fields. It has a quality character controlled by a pair of recessive genes (nuclear gene). The partial loss of Chl b in content affects the efficiency of light harvest in a light harvest complex (LHC), thus producing the difference of the exciting energy transfer and the efficiency of photochemistry conversion between the mutant and wild-type rice in photosynthetic unit. The efficiency of utilizing light energy is higher in the mutant than that in the wildtype rice relatively. For further discussion of the above-mentioned difference and learning about the mechanism of the increase in the photochemical efficiency of the mutant, the pico-second resolution fluorescence spectrum measurement with delay-frame-scanning single photon counting technique is adopted. Thylakoid membranes of the mutant and the wild-type rice are excited by an Ar+ laser with a pulse width of 120 ps, repetition rate of 4 MHz and wavelength of 514 nm. Compared with the time and spectrum property of exciting fluorescence, conclusions of those ultrafast dynamic experiments are: 1) The speeds of the exciting energy transferred in photo-system I are faster than that in photo-system II in both samples. 2) The speeds of the exciting energy transfer of mutant sample are faster than those of the wildtype. This might be one of the major reasons why the efficiency of photosynthesis is higher in mutant than that in the wild-type rice.

Characteristics and comparative study of chlorophyll-protein complexes from siphonous green algae

By mild PAGE method, 11, 11, 7 and 9 chlorophyll-protein complexes were isolated from two species of siphonous green algae ( Codium fragile (Sur.) Harlot and Bryopsis corticulans Setch.), green alga (Ulothrix flacca (Dillw.) Thur.), and spinach (Spinacia oleracea Mill.), respectively. Apparent molecular weights, Chi a/b ratios, distribution of chlorophyll, absorption spectra, low temperature fluorescence spectra of these complexes were determined, and compared with one another. PS I complexes of two siphonous green algae are larger in apparent molecular weight because of the attachment of relative highly aggregated LHC I. Four isolated light-harvesting complexes of PSII are all siphonaxanthin-Chl a/b-protein complexes, and they are not monomers and oligomers like those in higher plants. Especially, the absence of 730 nn fluorescence in PS I complexes indicates a distinct structure and energy transfer pattern.

Fluorescence Characterization of Dissolved Organic Matter in the East China Sea after Diatom Red Tide Dispersion

Fluorescence excitation-emission spectroscopy (EEMS) was employed to analyze the 3-dimensional fluorescence of dissolved organic matter in the East China Sea after diatom red tide dispersion. The relationships between fluorescence peak intensity, and salinity and chlorophyll-a were discussed. The centers of protein-like fluorescence peaks dispersed at Ex(max)/Em(max) = 270-280/290-315 nm (Peak B), 220-230/290-305 nm (Peak D), 230-240/335-350 nm(Peak S)and 280/320 nm(Peak T). Two humic-like peaks appeared at 255-270/435-480 nm (Peak A) and 330-350/420-480 nm(Peak C). High tyrosine-like intensity was observed in diatom red tide dispersion area, and tryptophan-like fluorescence was also found which was lower. High FIB/FIS showed that diatom red tide produced much tyrosine-like matter during dispersion. Peaks S, A and C had positive correlation with one another, and their distributions were similar, which decreased with distance increasing away from the shore. Good negative correlations between peaks S, A and C and salinity suggested that Jiangsu-Zhejiang coastal water was the same source of then-L Correlations between fluorescence peak intensity and chlorophyll-a were not remarkable enough to clear the relationship between fluorescence and living algal matter. It was supposed that the living algal matter contributed little to the fluorescence intensity of algal dispersion seawater.

Estimating Oceanic Primary Production Using Vertical Irradiance and Chlorophyll Profiles from Ocean Gliders in the North Atlantic

An autonomous underwater vehicle (Seaglider) has been used to estimate marine primary production (PP) using a combination of irradiance and fluorescence vertical profiles. This method provides estimates for depth-resolved and temporally evolving PP on fine spatial scales in the absence of ship-based calibrations. We describe techniques to correct for known issues associated with long autonomous deployments such as sensor calibration drift and fluorescence quenching. Comparisons were made between the Seaglider, stable isotope (13C), and satellite estimates of PP. The Seaglider-based PP estimates were comparable to both satellite estimates and stable isotope measurements.

Use of a fluorescence-based approach to assess short-term responses of the alga Pseudokirchneriella subcapitata to metal stress

This work explores the use of fluorescent probes to evaluate the responses of the green alga Pseudokirchneriella subcapitata to the action of three nominal concentrations of Cd(II), Cr(VI), Cu(II) and Zn(II) for a short time (6 h). The toxic effect of the metals on algal cells was monitored using the fluorochromes SYTOX Green (SG, membrane integrity), fluorescein diacetate (FDA, esterase activity) and rhodamine 123 (Rh123, mitochondrial membrane potential). The impact of metals on chlorophyll a (Chl a) autofluorescence was also evaluated. Esterase activity was the most sensitive parameter. At the concentrations studied, all metals induced the loss of esterase activity. SG could be used to effectively detect the loss of membrane integrity in algal cells exposed to 0.32 or 1.3 μmol L−1 Cu(II). Rh123 revealed a decrease in the mitochondrial membrane potential of algal cells exposed to 0.32 and 1.3 μmol L−1 Cu(II), indicating that mitochondrial activity was compromised. Chl a autofluorescence was also affected by the presence of Cr(VI) and Cu(II), suggesting perturbation of photosynthesis. In conclusion, the fluorescence-based approach was useful for detecting the disturbance of specific cellular characteristics. Fluorescent probes are a useful diagnostic tool for the assessment of the impact of toxicants on specific targets of P. subcapitata algal cells.

Chlorophyll a spatial inference using artificial neural network from multispectral images and in situ measurements

Considering the importance of monitoring the water quality parameters, remote sensing is a practicable alternative to limnological variables detection, which interacts with electromagnetic radiation, called optically active components (OAC). Among these, the phytoplankton pigment chlorophyll a is the most representative pigment of photosynthetic activity in all classes of algae. In this sense, this work aims to develop a method of spatial inference of chlorophyll a concentration using Artificial Neural Networks (ANN). To achieve this purpose, a multispectral image and fluorometric measurements were used as input data. The multispectral image was processed and the net training and validation dataset were carefully chosen. From this, the neural net architecture and its parameters were defined to model the variable of interest. In the end of training phase, the trained network was applied to the image and a qualitative analysis was done. Thus, it was noticed that the integration of fluorometric and multispectral data provided good results in the chlorophyll a inference, when combined in a structure of artificial neural networks.

Das wasserlösliche Chlorophyll-Protein (WSCP): biochemische Charakterisierung und Untersuchungen zur biologischen Funktion

Das WSCP (water-soluble chlorophyll protein) der Brassicaceen ist das einzig bekannte Chlorophyll-bindende Protein, welches keine Carotinoide bindet. Es ist ein wasserlösliches, ca. 80 kDa großes Homotetramer mit 1-4 gebundenen Chlorophyllen. Das Protein ist äußerst stabil und vermag die gebundenen Chlorophylle vor Photooxidation zu schützen. Seine Funktion in der Pflanze ist bis heute ein Rätsel und sollte in dieser Arbeit zusammen mit seinen biochemischen Eigenschaften weiter aufgeklärt werden. Es wurden Versuche durchgeführt mit nativem und rekombinantem WSCP aus Blumenkohl (BoWSCP bzw. BoWSCPhis) und aus Arabidopsis thaliana (AtWSCP bzw. AtWSCPhis). Die Expressionsausbeute von BoWSCPhis konnte verbessert werden und zusätzlich wurde die Rekonstitutionsmethode für das rekombinante WSCP optimiert, sodass das pigmentierte Protein mit hoher Ausbeute und großer Reinheit gewonnen werden konnte. Zudem wurde ein neuer WSCP-Klon hergestellt, mBoWSCPhis, der in seiner Sequenz dem maturen nativen BoWSCP entspricht und weitaus weniger Aggregationsprobleme zeigte als BoWSCPhis. Weiterführende Versuche zur Stabilität und dem Oligomerisierungsgrad von WSCP haben die neue Erkenntnis erbracht, dass die Phytolschwänze der von WSCP gebundenen Chlorophylle zwar essentiell sind für die Stabilität von WSCP-Oligomeren, nicht aber für die Oligomerisierung selbst, wie es in der Literatur bislang postuliert wurde. Zusätzlich zu ihrer außerordentlichen Hitzestabilität erwiesen sich die Chl-WSCP-Komplexe als stabil in einem breiten pH-Spektrum. AtWSCPhis besaß eine vergleichbare Stabilität, und auch das Oligomerisierungsverhalten zeigte Ähnlichkeiten zu BoWSCPhis. Im Rahmen einer Forschungskooperation mit dem Institut für Optik und Atomare Physik der TU Berlin wurden zeitaufgelöste Absorptionsspektren sowie Tieftemperatur-Fluoreszenzspektren an Chl-WSCP-Komplexen gemessen. Die Ergebnisse zeigten deutlich, dass die WSCP-gebundenen Chlorophylle excitonisch gekoppelt sind und wiesen zudem auf unterschiedliche Chl-Bindungsmodi hin. Aufgrund seines einfachen Aufbaus und seines geringen Chlorophyllgehalts hat sich WSCP bei diesen Versuchen als sehr geeignetes Modellsystem erwiesen, um Messungen zur Chlorophyllbindung mit Vorhersagen aus theoretischen Modellen zu vergleichen. Bei den Experimenten zur biologischen Funktion wurden einerseits Arabidopsis thaliana WSCP-„knock-out“-Pflanzen unter verschiedenen Bedingungen charakterisiert, andererseits wurden Experimente mit rekombinantem WSCP durchgeführt, um eine mögliche Interaktion mit anderen Proteinen zu detektieren. Die vegetativen Stadien der Mutante zeigten keinen Phänotyp; das native Arabidopsis-WSCP konnte später bei der Wildtyp-Pflanze ausschließlich in jungen Schoten lokalisiert werden, was eine Erklärung hierfür lieferte. Rekombinantes WSCP konnte Chlorophylle aus nativem LHCII entfernen, eine Interaktion mit Chlorophyllase konnte jedoch nicht nachgewiesen werden; daher konnte auch die Hypothese, WSCP sei ein Chl-Carrier beim Chl-Abbau, nicht untermauert werden. Bei den durchgeführten Enzym-Assays wurde eine geringfügige Inhibition der Cysteinprotease Papain beobachtet, aber keine Inhibition der Serinprotease Trypsin, obwohl Blumenkohl-WSCP N-proximal das Motiv der Künitz-Proteaseinhibitoren besitzt. Die Frage nach der biologischen Funktion von WSCP bleibt also weiterhin offen.