Sample preconditioning for measurement of fluorescence induction of chlorophyll a in marine phytoplankton

Conditions for measuring fluorescence induction curves (time-scalems) of in vivo chlorophyll ^a were studied using cultures ofDunaliella tertiolecta Butcher (Chlorophyceae) and of Thalassiosirapseudonana Hustedt (3H) (Bacillariophyceae), and samples ofnatural phytoplankton populations from the Grand Banks. Thearea above the fluorescence induction curve (A_DCMU) and themaximum fluorescence intensity (F_max) measured in the presenceof 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU) were computedby microcomputer. Cells must be ‘conditioned’ or‘adapted’ prior to obtaining a fluorescence inductioncurve; dark-adaptation resulted in a lower A_DCMU and F_max thandid adaptation in far-red (720 nm) light, and was the conditioningmethod chosen. A_DCMU and F_max increased linearly with increasingirradiance up to 32.8 W m^–2 the highest actinic irradianceavailable. Information on the light history of D. tertiolectawas obtained by following the time-course of change in A_DCMUand in F_max for cells exposed for 10 min to far-red or to bluelight. The rise-time of the fluorescence induction curve andvalues of F_max were greater for samples of D. tertiolecta concentratedonto glass-fiber filters than for liquid samples, however, valuesof A_DCMU for filtered and liquid samples were not significantlydifferent. Samples of Grand Banks phytoplankton collected ontoglass-fiber filters and frozen for 28 d exhibited a significantdecrease in F_max and in A_DCMU relative to the same freshly-filteredsamples. Filtration and freezing of samples is not recommended. ^*This paper is the result of a study made at the Group for AquaticPrimary Productivity (GAP). Second International Workshop heldat the National Oceanographic Institute. Haifa. Israel in April–May1984.     

In vivo fluorescence excitation and absorption spectra of marine phytoplankton: I. Taxonomic characteristics and responses to photoadaptation

Absorption and fluorescence excitation spectra were measuredfor batch cultures of five species of marine phytoplankton grownunder high and low light. These spectra were examined for propertiescharacteristic of taxonomic position and of photoadaptive response.While regions of absorption and excitation of chlorophyll afluorescence diagnostic of pigment composition were identifiable,photoadaptive response had greater influence on spectral variability.Although reduced growth irradiance caused changes in both theabsorption and fluorescence excitation spectra, the fluorescenceexcitation spectrum appears to be more sensitive to alterationsin the ambient light field for growth than does the absorptionspectrum. For a single species. the fluorescence excitationspectrum for a sample grown at low irradiance showed greaterstructure than that for the sample grown at a high irradiance.Under low light conditions, the excitation of chlorophyll afluorescence by accessory pigments increased relative to theexcitation by chlorophyll a itself The highest fluorescenceyields occur in the blue-green region of the spectrum, correspondingto bands of peak absorption by the accessory pigments. Changesin absorption spectra are less marked, but two features recur.First. in the blue-green region of the spectrum from -500–560nm. absorption is enhanced in the low-light cells relative tothat of the high-light cells. Second, the ratio of absorptionat 435 nm to that at 676 nm was greater for the high-light cells.Correlating changes in pigment concentrations were observed.The influence of photoadaptation on the properties of fluorescenceexcitation spectra is as great or greater than the influenceof pigment complements characteristic of specific algal taxa.     

Variability in chlorophyll a specific absorption coefficient in marine phytoplankton as a function of cell size and irradiance

Size-dependence of chlorophyll a (Chl a) specific absorptioncoefficient a*(     

Enzymatic degradation of chlorophyll a by marine phytoplankton in vitro

The enzymatic degradation of chlorophyll a and the formation of chlorophyllide a, phaeophytin a, and phaeophorbide a were detected in vitro in several species of marine phytoplankton. Loss of phytol and Mg²⁺ were found to be catalysed by chlorophyllase and a magnesium-releasing enzyme, respectively. The activities of the two enzymes could be distinguished from each other by inhibiting with Mg²⁺ and/or p-chloromercurobenzoate. Both enzymes are activated by cell disintegration. Degradation products were not detected spectrophotometrically in vivo. Additionally, in some species, chlorophyll a was degraded to products which do not absorb visible light.     

The use of spectral fluorescence methods to detect changes in the phytoplankton community

In vivo fluorescence methods are efficient toolsfor studying the seasonal and spatial dynamics ofphytoplankton. Traditionally the measurements are madeusing single excitation-emission wavelengthcombination. During a cruise in the Gulf of Riga(Baltic Sea) we supplemented this technique bymeasuring the spectral fluorescence signal (SFS) andfixed wavelength fluorescence intensities at theexcitation maxima of main accessory pigments. Thesemethods allowed the rapid collection of quantitativefluorescence data and chemotaxonomic diagnostics ofthe phytoplankton community. The chlorophylla-specific fluorescence intensities (R) and thespectral fluorescence fingerprints were analysedtogether with concentrations of chlorophyll a indifferent algal size-groups, phytoplankton biomass andtaxonomic position. The lower level of R in thesouthern gulf was related to the higher proportion ofcyanobacteria relative to total biomass and the lowerabundance of small algae. The phycoerythrinfluorescence signal was obviously due to the largecyanobacteria. The basin-wide shift in the shape ofchlorophyll a excitation spectra was caused bythe variable proportions of differently pigmentedcyanobacteria, diatoms and cryptomonads. This revised version was published online in July 2006 with corrections to the Cover Date.     

Corrected fluorescence excitation and emission spectra of phytoplankton: toward a more uniform approach to fluorescence measurements

Techniques for correction of fluorescence emission and excitationspectra of phytoplankton are described, which can be appliedin any commercially available spectrophotometer. The correctionof the emission spectrum is based on the measurement of a calibratedlight source. The excitation spectra are corrected by meansof a quantum counter solution that measures the spectral intensityof the excitation system and separate correction for wavelength-dependenteffects of the excitation optics. The correction proceduresgive technically corrected spectra, i.e. spectra that are freefrom wavelength dependent bias, but do not give absolute intensityvalues. Spectra that have been properly corrected for instrumentalwavelength dependencies are suitable for intercomparison, bothintra- and interlaboratory. Another application is the derivationof spectral data that will be obtained by other techniques thatmake use of fluorescence measurements, such as flow cytometry,remote sensing and in situ instruments. A necessary conditionis that the spectral response functions of these instrumentsmust be known. ¹Present address: AKZO, Arla-CRL, PO Box 9300, NL-6800 SB Arnhem,The Netherlands     

Photoadaptation of two members of the Chlorophyta (Scenedesmus and Chlorella) in laboratory and outdoor cultures: changes in chlorophyll fluorescence quenching and the xanthophyll cycle

The role of the xanthophyll cycle in the adaptation of two chlorococcal algae Scenedesmus quadricauda and Chlorella sorokiniana to high irradiance was studied under laboratory and outdoor conditions. We wished to elucidate whether the xanthophyll cycle plays a key role in dissipating the excesses of absorbed light, as in higher plants, and to characterise the relationship between chlorophyll fluorescence parameters and the content of xanthophyll-cycle pigments. The xanthophyll cycle was found to be operative in both species; however, its contribution to overall non-photochemical quenching (NPQ) could only be distinguished in Scenedesmus (15–20% of total NPQ). The Scenedesmus cultures showed a larger pool of xanthophyll-cycle pigments than Chlorella, and lower sensitivity to photoinhibition as judged from the reduction of maximum quantum yield of photosystem II. In general, both algae had a larger xanthophyll-cycle pool when grown outdoors than in laboratory cultures. Comparing the two species, Scenedesmus exhibited a higher capacity to adapt to high irradiance, due to an effective quenching mechanism and high photosynthetic capacity; in contrast, Chlorella represents a species with a larger antennae system, less-efficient quenching and lower photosynthetic performance. Non-photochemical quenching (NPQ) induced through the xanthophyll cycle can, to a limited extent, represent a regulatory factor in diluted algal cultures grown in outdoor solar photobioreactors, as well as in natural algal phytoplankton populations exposed transiently to high irradiance. However, it does not play an appreciable role in dense, well-mixed microalgal suspensions. Received: 6 August 1998 / Accepted: 12 February 1999     

Two-photon excitation chlorophyll fluorescence lifetime imaging: a rapid and noninvasive method for in vivo assessment of cadmium toxicity in a marine diatom Thalassiosira weissflogii

We demonstrate that a two-photon excitation fluorescence lifetime imaging technology can rapidly and noninvasively assess the cadmium (Cd)-induced toxic effects in a marine diatom Thalassiosira weissflogii. The chlorophyll, an intrinsic fluorophore, was used as a contrast agent for imaging of cellular structures and for assessment of cell toxicity. The assessment is based on an imaging-guided statistical analysis of chlorophyll fluorescence decay. This novel label-free imaging method is physically based and free of tedious preparation and preprocessing of algal samples. We first studied the chlorophyll fluorescence quenching induced by the infrared two-photon excitation laser and found that the quenching effects on the assessment of Cd toxicity could be well controlled and calibrated. In the toxicity study, chlorophyll fluorescence lifetime images were collected from the diatom samples after exposure to different concentrations of Cd. The alteration of chloroplast structure at higher Cd concentration was clearly identified. The decay of chlorophyll fluorescence extracted from recorded pixels of high signal-to-noise ratio in the fluorescence lifetime image was analyzed. The increase of average chlorophyll fluorescence lifetime following Cd treatment was observed, indicating the Cd inhibition effect on the electron transport chain in photosynthesis system. The findings of this study show that the temporal characteristics of chlorophyll fluorescence can potentially be utilized as a biomarker for indicating Cd toxicity noninvasively in algal cells.     

Profiles of light absorption and chlorophyll within spinach leaves from chlorophyll fluorescence

Chlorophyll fluorescence was used to estimate profiles of absorbed light within chlorophyll solutions and leaves. For chlorophyll solutions, the intensity of the emitted fluorescence declined in a log–linear manner with the distance from the irradiated surface as predicted by Beer's law. The amount of fluorescence was proportional to chlorophyll concentration for chlorophyll solutions given epi‐illumination on a microscope slide. These relationships appeared to hold for more optically complex spinach leaves. The profile of chlorophyll fluorescence emitted by leaf cross sections given epi‐illumination corresponded to chlorophyll content measured in extracts of leaf paradermal sections. Thus epifluorescence was used to estimate relative chlorophyll content through leaf tissues. Fluorescence profiles across leaves depended on wavelength and orientation, reaching a peak at 50–70 µm depth. By infiltrating leaves with water, the pathlengthening due to scattering at the airspace : cell wall interfaces was calculated. Surprisingly, the palisade and spongy mesophyll had similar values for pathlengthening with the value being greatest for green light (550 > 650 > 450 nm). By combining fluorescence profiles with chlorophyll distribution across the leaf, the profile of the apparent extinction coefficient was calculated. The light profiles within spinach leaves could be well approximated by an apparent extinction coefficient and the Beer–Lambert/Bouguer laws. Light was absorbed at greater depths than predicted from fibre optic measurements, with 50% of blue and green light reaching 125 and 240 µm deep, respectively.     

Light-induced changes of NADPH fluorescence in isolated chloroplasts: a spectral and fluorescence lifetime study

Isolated chloroplasts show a light-induced reversible increase in blue-green fluorescence (BGF), which is only dependent on NADPH changes. In the present communication, we report a time-resolved and spectral analysis of this BGF in reconstituted chloroplasts and intact isolated chloroplasts, in the dark and under actinic illumination. From these measurements we deduced the contribution of the different forms of NADPH (free and bound to proteins) to the light-induced variation of BGF and conclude that this variation is due only to the redox change of the NADP pool. A simple model estimating the distribution of NADPH between the free and bound form was designed, that explains the differences measured for the BGF of reconstituted chloroplasts and intact chloroplasts. From the decay-associated spectra of the chloroplast BGF, we also deduced the participation of flavins to the green peak of chloroplast fluorescence emission spectrum, and the existence of excitation energy transfer from proteins to bound NADPH in chloroplasts. In addition, we re-examined the use of chloroplast BGF as a quantitative measure of NADPH concentration, and confirmed that chloroplast BGF can be used for non-destructive, continuous and probably quantitative monitoring of light-induced changes in NADP redox state.     

Relevance of various formulations of phytoplankton chlorophyll a:carbon ratio in a 3D marine ecosystem model

Numerous experimental studies showed that the phytoplankton Chla-to-Carbon ratio (Chla:C) is highly variable, whereas most of the marine ecosystem models use a constant ratio. In this work, we tested three different formulations for computing the modelled Chla in a 3D coupled hydrodynamical-biogeochemical model of the Southwest lagoon of New Caledonia. The first formulation considers a constant Chla:C ratio. In the second one, Chla is a diagnostic variable related to the variable phytoplankton nitrogen-to-carbon ratio. In the last formulation, Chla is a state variable of the model, which is dynamically simulated. Results showed important differences between the formulations, the first leading to overestimate the Chla concentration in low nutrients conditions. Thus, this study strengthens the importance of the Chla modelling in a coupled model in order to better estimate a crucial variable for validation of ecosystem models.     

Lettuce flavonoids screening and phenotyping by chlorophyll fluorescence excitation ratio

Main conclusion

Environmentally induced variation and the genotypic differences in flavonoid and phenolic content in lettuce can be reliably detected using the appropriate parameters derived from the records of rapid non-invasive fluorescence technique.

The chlorophyll fluorescence excitation ratio method was designed as a rapid and non-invasive tool to estimate the content of UV-absorbing phenolic compounds in plants. Using this technique, we have assessed the dynamics of accumulation of flavonoids related to developmental changes and environmental effects. Moreover, we have tested appropriateness of the method to identify the genotypic differences and fluctuations in total phenolics and flavonoid content in lettuce. Six green and two red genotypes of lettuce (Lactuca sativa L.) grown in pots were exposed to two different environments for 50 days: direct sunlight (UV-exposed) and greenhouse conditions (low UV). The indices based on the measurements of chlorophyll fluorescence after red, green and UV excitation indicated increase of the content of UV-absorbing compounds and anthocyanins in the epidermis of lettuce leaves. In similar, the biochemical analyses performed at the end of the experiment confirmed significantly higher total phenolic and flavonoid content in lettuce plants exposed to direct sun compared to greenhouse conditions and in red compared to green genotypes. As the correlation between the standard fluorescence indices and the biochemical records was negatively influenced by the presence of red genotypes, we proposed the use of a new parameter named Modified Flavonoid Index (MFI) taking into an account both absorbance changes due to flavonol and anthocyanin content, for which the correlation with flavonoid and phenolic content was relatively good. Thus, our results confirmed that the fluorescence excitation ratio method is useful for identifying the major differences in phenolic and flavonoid content in lettuce plants and it can be used for high-throughput pre-screening and phenotyping of leafy vegetables in research and breeding applications towards improvement of vegetable health effects.

     

The presence of chlorophyll b and the estimation of phaeopigments in marine phytoplankton

A reverse-phase h.p.l.c. technique was used to estimate theconcentration of chlorophyll b in phytoplankton cultures, fecalpellets of Calanus pacificus, and suspended paniculate matterfrom the Central North Pacific, Oregon coastal waters, and DabobBay (a temperate fjord in Puget Sound, WA, USA). The purposewas to assess the distribution of this pigment in the euphoticzone and its effect on the fluorometnc estimation of phaeopigments.Analyses of natural waters confirm high chlorophyll b concentrations(median mass ratio of b:a > 0.3) at the depth of the chlorophylla maximum in tropical waters while values for temperate planktonare relatively low (median mass ratio of chl b:a = 0.05) andpatchy. Zooplankton fecal pellets showed a significant enrichmentin chlorophyll b, suggesting grazing as a mechanism to explainhigh concentrations of this pigment at the bottom of the euphoticzone. It is estimated that the presence of chlorophyll b couldcause an average overestimation of phaeopigment concentrationby the fluorometnc technique of 38% between 0 and 200 m in theCentral North Pacific. This effect is more pronounced at thelayer of chlorophyll b maximum (120–140 m). ¹Present address: Marine Biology Research Division, A-002, ScrippsInstitution of Oceanography, La Jolla, CA 92093, USA     

Determination of chlorophylls and carotenoids of marine phytoplankton: separation of chlorophyll a from divinylchlorophyll a and zeaxanthin from lutein

A rapid reverse-phase HPLC method is presented for the identificationand quantification of most of the phytoplankton pigments. Thismethod yields the resolution of divinyl-chlorophyll a and chlorophylla, as well as the partial resolution of lutein and zeaxanthin,and of divinyl-chlorophyll b and chlorophyll b. In addition,chlorophylls c_1,2 and c₃ are well resolved. The analysis timefor one sample is 20 mm, which makes this method particularlysuited when large numbers of samples have to be processed.     

Dehydration-induced changes in spectral reflectance indices and chlorophyll fluorescence of Antarctic lichens with different thallus color,and intrathalline photobiont

In this study, we investigated responses of the Photochemical Reflectance Index (PRI), and Normalized Difference Vegetation Index (NDVI) to gradual dehydration of several Antarctic lichen species (chlorolichens: Xanthoria elegans, Rhizoplaca melanophthalma, Physconia muscigena, cyanolichen: Leptogium puberulum), and a Nostoc commune colony from fully wet to a dry state. The gradual loss of physiological activity during dehydration was evaluated by chlorophyll fluorescence parameters. The experimental lichen species differed in thallus color, and intrathalline photobiont. In the species that did not exhibit color change with desiccation (X. elegans), NDVI and PRI were more or less constant (mean of 0.25, ??0.36, respectively) throughout a wide range of thallus hydration status showing a linear relation to relative water content (RWC). In contrast, the species with apparent species-specific color change during dehydration exhibited a curvilinear relation of NDVI and PRI to RWC. PRI decreased (R. melanophthalma, L. puberulum), increased (N. commune) or showed a polyphasic response (P. muscigena) with desiccation. Except for X. elegans, a curvilinear relation was found between the NDVI response to RWC in all species indicating the potential of combined ground research and remote sensing spectral data analyses in polar regions dominated by lichen flora. The chlorophyll fluorescence data recorded during dehydration (RWC decreased from 100 to 0%) revealed a polyphasic species-specific response of variable fluorescence measured at steady state—F_s, effective quantum yield of photosystem II (Φ_PSII), and non-photochemical quenching (qN). Full hydration caused an inhibition of Φ_PSII in N. commune while other species remained unaffected. The dehydration-dependent fall in Φ_PSII was species-specific, starting at an RWC range of 22–32%. Critical RWC for Φ_PSII was around 5–10%. Desiccation led to a species-specific polyphasic decrease in F_s and an increase in qN indicating the involvement of protective mechanisms in the chloroplastic apparatus of lichen photobionts and N. commune cells. In this study, the spectral reflectance and chlorophyll fluorescence data are discussed in relation to the potential of ecophysiological processes in Antarctic lichens, their resistance to desiccation and survival in Antarctic vegetation oases.     

Correlation between monovalent cation-induced decreases in chlorophyll a fluorescence and chloroplast structural changes

Low concentrations (~ 3 mm) of salts of monovalent cations such as Na⁺, K⁺, and tetraethylammonium were found to decrease the turbidity of chloroplast suspensions. The turbidity changes (Δ₅₄₀) had the same kinetics, salt concentration dependence, and pH dependence as the monovalent cation-induced decreases in chlorophyll a fluorescence (9), suggesting that structural changes are the cause of the associated increases in spillover. Electron microscopy revealed that the grana are stacked when spillover is inhibited (in the absence of salts or the presence of divalent cations) and that monovalent cations cause the grana to unstack, thereby promoting spillover.     

The chlorophyll fluorescence quenching and changes of absorbance in pea chloroplasts

Chlorophyll (Chl) fluorescence quenching parameters were measured in dark-adapted pea leaves and chloroplasts with the purpose to find the conditions of high and low non-photochemical quenching, that would be stable during a prolonged irradiation. A PAM fluorometer was used for measuring induction curves in the range of actinic radiation of 3-35 W m-2, with an ordinary value of about 15 W m-2. The effects of various mediators, i.e., ascorbate, methyl viologen (MV), dithiothreitol (DTT) and nigericin, on the quenching process were tested. Simultaneously, the absorbance was measured during a 15-20 min period of irradiation and after the actinic radiation was turned off, i.e., in the recovery period. The pH values of chloroplast suspensions were 5.5, 6.5 and 8.0, the largest non-photochemical quenching was observed at pH of 6.5. The irradiation of chloroplasts led to an absorption decrease within the entire photosynthetically active range, attaining saturation when the fluorescence reached Fs level, and to an absorption increase during the recovery period. Absorbance changes at the maximum of red band were 10-20 %. A decrease in Chl concentration (10 %) after irradiation was found only at pH of 5.5, when the recovery time was the longest, i.e., about 60 min. This revised version was published online in June 2006 with corrections to the Cover Date.     

The chlorophyll fluorescence quenching and changes of absorbance in pea chloroplasts

Chlorophyll (Chl) fluorescence quenching parameters were measured in dark-adapted pea leaves and chloroplasts with the purpose to find the conditions of high and low non-photochemical quenching, that would be stable during a prolonged irradiation. A PAM fluorometer was used for measuring induction curves in the range of actinic radiation of 3-35 W m-2, with an ordinary value of about 15 W m-2. The effects of various mediators, i.e., ascorbate, methyl viologen (MV), dithiothreitol (DTT) and nigericin, on the quenching process were tested. Simultaneously, the absorbance was measured during a 15-20 min period of irradiation and after the actinic radiation was turned off, i.e., in the recovery period. The pH values of chloroplast suspensions were 5.5, 6.5 and 8.0, the largest non-photochemical quenching was observed at pH of 6.5. The irradiation of chloroplasts led to an absorption decrease within the entire photosynthetically active range, attaining saturation when the fluorescence reached Fs level, and to an absorption increase during the recovery period. Absorbance changes at the maximum of red band were 10-20 %. A decrease in Chl concentration (10 %) after irradiation was found only at pH of 5.5, when the recovery time was the longest, i.e., about 60 min.