Extraction of chlorophyll and carotenoid pigments from Antarctic benthic mats for analysis by HPLC

The efficiency of 9:1 acetone-water, DMSO and boiling 9:1 ethanol-water in extracting chlorophyll and carotenoid pigments from benthic cyanobacterial mats from Antarctica for HPLC (high performance liquid chromatography) analysis was examined. Considerable breakdown of chlorophylla was observed after 5 min extraction in boiling ethanol and 2 h extraction in DMSO. Over 50% of the chlorophylla was degraded to chlorophyllidea and there was substantial loss of carotenoids after a 15 h exposure of ground cells to cold 9:1 acetone-water.Mild sonication of ground mat material in 9:1 acetone-water followed by a 4 h extraction at 4 ° C was found to minimise chlorophylla breakdown and dramatically improved the extraction efficiency of chlorophylla, myxoxanthophyll and -carotene.     

A mathematical model for the vertical distribution of chlorophyllA in estuarine intertidal sediments

Coastal and estuarine intertidal sediments are commonly colonized by dense populations of microphytobenthos. Due to wind and tides, important fractions of microphytobenthic populations may be buried. A mathematical model describing the depth variation of chlorophyll a in intertidal sediments was developed and experimentally tested. The model assumed first-order chlorophylla degradation and a constant mean burial velocity which resulted in a negative exponential variationC _Z =C _O exp{-k/vz} (C _Z andC _O =chlorophylla concentration at depth zand at the surface;k=specific degradation rate of chlorophyll a to pheopigments;V=mean burial velocity). Chlorophylla concentration depth profiles in different sediment types measured at the Tagus estuary and Ria Formosa (Portugal) were used to validate the model. The model was adjusted to field data. The chlorophyll a degradation rate was measured in a microcosm experiment under total darkness and no tidal action, and sampled during three months. This rate was shown to be independent of time and depth for the upper 0–15 mm depth interval. This result allowed the estimation ofV for each sampling site. Comparison of predicted and observed temporal data further confirmed the validity of the model andk andV values. Despite its simplicity, the proposed model adequately described the depth distribution of chlorophylla in different types of intertidal sediments. The model allowed the quantitative characterization of the buried microphytobenthic biomass (depth-integrated biomass) and the assessment of its importance as potentially productive stock of cells.     

Pigment composition of putatively achlorophyllous angiosperms

Chlorophyll and carotenoid pigment composition was determined for ten species of putatively achlorophyllous angiosperms using high-performance liquid chromatography. Four families were represented:Lennoaceae (Pholisma arenarium);Monotropaceae (Allotropa virgata, Monotropa uniflora, Pterospora andromedea, Sarcodes sanguinea); Orobanchaceae (Epifagus virginiana, Orobanche cooperi, O. uniflora);Orchidaceae (Cephalanthera austinae, Corallorhiza maculata). Chlorophylla was detected in all taxa, but chlorophyllb was only detected inCorallorhiza maculata. The relative amount of chlorophyll and chlorophyll-related pigments in these plants is greatly reduced compared to fully autotrophic angiosperms.     

Domain structures of chlorophyllide<Emphasis Type="Italic"> a</Emphasis> oxygenase of green plants and<Emphasis Type="Italic"> Prochlorothrix hollandica</Emphasis> in relation to catalytic functions

Chlorophyll b is a photosynthetic antenna pigment found in prochlorophytes and chlorophytes. In chlorophytes, its biosynthesis regulates the photosynthetic antenna size. Chlorophyll b is synthesized from chlorophyll a in a two-step oxygenation reaction by chlorophyllide a oxygenase (CAO). In this study, we first identified the entire sequence of a prochlorophyte CAO gene from Prochlorothrix hollandica to compare it with those from chlorophytes, and we examined the catalytic activity of the gene product. Southern blot analysis showed that the CAO gene is presented in one copy in the P. hollandica genome. The P. hollandica CAO gene (PhCAO) has a coding capacity for 367 amino acids, which is much smaller than that of Arabidopsis thaliana (537 amino acids) and Oryza sativa (542 amino acids) CAO genes. In spite of the small size, PhCAO catalyzed the formation of chlorophyll b. By comparing these sequences, we classified the land-plant sequences into four parts: the N-terminal sequence predicted to be a transit peptide, the successive conserved sequence unique in land plants (A-domain, 134 amino acids), a less-conserved sequence (B-domain, 30 amino acids) and the C-terminal conserved sequence common in chlorophytes and prochlorophytes (C-domain, 337 to 344 amino acids). We demonstrated that the C-domain is sufficient for catalytic activity by transforming the cyanobacterium Synechocystis sp. PCC6803 with the C-domain from A. thaliana. In this paper, the role of the A-domain is discussed in relation to the formation of light-harvesting chlorophyll a/b–protein complexes in land plants.Abbreviations CAO Chlorophyllide a oxygenase - CP Chlorophyll protein - HPLC High-performance liquid chromatography - LHC Light-harvesting complex - PCR Polymerase chain reaction - PS Photosystem     

Detecting presence of phytoplankton groups with non-specific pigment signatures

The use of pigment ratios and sizefractionation was investigated fordetecting the presence of phytoplanktongroups with non-specific pigmentsignatures. These groups includeprymnesiophytes, chlorophytes,prasinophytes and cyanobacteria. Prasinophytes without the diagnosticpigment prasinoxanthin could be identifiedby calculating ratios ofprasinoxanthin/chlorophyll b andlutein/chlorophyll b on data setsfrom two Danish estuaries. Furthermore,these pigment ratios could be used toexclude the presence of chlorophytes, andratios of zeaxanthin/chlorophyll brevealed that cyanobacteria wereoccasionally present. This information isimportant when selecting whichphytoplankton groups to load in the CHEMTAXprogram for calculating the chlorophylla biomass of the individualphytoplankton groups. Subtypes ofprymnesiophytes were detected by sizefractionations on samples from a mesocosmexperiment. The size fractionations,although time-consuming and not alwaysfeasible, and the pigment ratiocalculations proved to be able to providethe desired information as to whichphytoplankton groups to load in the CHEMTAXprogram.     

Trace determination of urinary 3-hydroxybenzo[a]pyrene by automated column-switching high-performance liquid chromatography

3-Hydroxybenzo[a]pyrene (3-OHB[a]P), one of the metabolites of benzo[a]pyrene (B[a]P), has been determined in human urine using an automated column-switching procedure. The hydrolysed biological sample is centrifuged just prior to being injected into a reusable precolumn loop, which is packed with a preparative phase and coupled on-line to a liquid chromatographic (LC) system. A rapid pre-treatment of the hydrolysed sample, consisting of a concentration and a crude clean-up, is performed on the precolumn. The analytes are then non-selectively desorbed with the LC eluent and the sample is cleaned again in three successive purification columns using the direct transfer or “heart-cut” technique. The pre-treatment does not exceed 3 min. and the entire analytical purification and separation procedure takes less than 30 min. Average 3-OHB[a]P recovery reaches 95% in the 1–50 ng/l range of urine, and the detection limit is 0.1 ng/l urine for a 3 ml injection of hydrolysed urine. The developed method was compared with a more time-consuming off-line method to analyse urines of B[a]P gavaged rats; the statistical treatment indicates that both methods are in agreement. The method was applied to purify and concentrate the urine samples of workers exposed and apparently unexposed to polycyclic aromatic hydrocarbons (PAHs).     

Vertical structure and photosynthetic activity of Lake Shira phytoplankton

This study was conducted to analyse vertical dynamics of phytoplankton distribution in Shira Lake during the summer stratification regime. From late June to September phytoplankton in Shira Lake were stratified with the maximum in the lower part of the thermocline, at a depth of 8–12 m, with a chlorophyll concentration up to 23 g and biomass up to 5 mg l^–1. Maxima of chlorophyll and biomass of cyanobacteria and green algae were in different layers. From June to September a major part of chlorophyll a was in green algae, while under ice – in cyanobacteria. The variable fluorescence proves high photosynthetic activity of algae in the depth assemblage. Epifluorescent analysis disclosed that additional light-harvesting pigments were better developed in cells from the depth maximum. The maximum of gross primary production calculated from fluorescence corresponded to the depth maximum of phytoplankton. Primary production over a season was 2.7 gO₂ m^–2. Formation mechanisms of the depth maximum of phytoplankton are discussed in this paper.     

Conversion of chlorophyll b to chlorophyll a precedes magnesium dechelation for protection against necrosis in Arabidopsis

Chlorophyll is a deleterious molecule that generates reactive oxygen species and must be converted to non‐toxic molecules during plant senescence. The degradation pathway of chlorophyll a has been determined; however, that of chlorophyll b is poorly understood, and multiple pathways of chlorophyll b degradation have been proposed. In this study, we found that chlorophyll b is degraded by a single pathway, and elucidated the importance of this pathway in avoiding cell death. In order to determine the chlorophyll degradation pathway, we first examined the substrate specificity of 7‐hydroxymethyl chlorophyll a reductase. 7‐hydroxymethyl chlorophyll a reductase reduces 7‐hydroxymethyl chlorophyll a but not 7‐hydroxymethyl pheophytin a or 7‐hydroxymethyl pheophorbide a. These results indicate that the first step of chlorophyll b degradation is its conversion to 7‐hydroxymethyl chlorophyll a by chlorophyll b reductase, although chlorophyll b reductase has broad substrate specificity. In vitro experiments showed that chlorophyll b reductase converted all of the chlorophyll b in the light‐harvesting chlorophyll a/b protein complex to 7‐hydroxymethyl chlorophyll a, but did not completely convert chlorophyll b in the core antenna complexes. When plants whose core antennae contained chlorophyll b were incubated in the dark, chlorophyll b was not properly degraded, and the accumulation of 7‐hydroxymethyl pheophorbide a and pheophorbide b resulted in cell death. This result indicates that chlorophyll b is not properly degraded when it exists in core antenna complexes. Based on these results, we discuss the importance of the proper degradation of chlorophyll b.     

Importance quantitative de différentes classes de taille phytoplanctoniques en milieu lacustre eutrophe

The distribution of three phytoplanktonic size fractions was studied in an eutrophic lake. Size fractionation experiments were performed using cell enumeration by inverted microscopy and chlorophyll a estimation. The results were also compared with the ATP content in the analysed algal fractions.The (1–12 µm) fraction represented only on average 5 % of the total biomass, when estimated by the cell enumeration method, but made up 26 % of the total chlorophyll a and 33 % of the total ATP. This discrepancy confirms that cell enumeration with an inverted microscope seriously underestimates the nanoplankton biomass.The (12–45 µm) fraction made up to 50% of the total biomass regardless the method of analysis used. The chlorophyll a and biovolume estimation in this fraction were positively correlated.The contribution of algal species with large cell size (45–160 µm) to the total algal biomass was higher (39%) when based on their biovolume estimation than when on the chlorophyll a (27%). The low ATP contribution of this fraction (17%) may be attributable to high percentage of dead cells.

     

Characterization of the cytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>6</Subscript><Emphasis Type="Italic">f</Emphasis> complex from marine green alga, <Emphasis Type="Italic">Bryopsis corticulans</Emphasis>

A pure, active cytochrome b ₆ f was isolated from the chloroplasts of the marine green alga, Bryopsis corticulans. To investigate and characterize this cytochrome b ₆ f complex, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), absorption spectra measurement and HPLC were employed. It was shown that this purified complex contained four large subunits with apparent molecular masses of 34.8, 24, 18.7 and 16.7 kD. The ratio of Cyt b ₆ to Cytf was 2.01 : 1. The cytochromeb ₆ f was shown to catalyze the transfer of 73 electrons from decylplastoquinol to plastocyanin–ferricyanide per Cyt f per second. α-Carotene, one kind of carotenoid that has not been found to present in cytochrome b ₆ f complex, was discovered in this preparation by reversed phase HPLC. It was different from β-carotene usually found in cytochrome b ₆ f complex. The configuration of the major α-carotene component was assigned to be 9-cis by resonance Raman spectroscopy. Different from the previous reports, the configuration of this α-carotene in dissociated state was determined to be all-trans. Besides this carotene, chlorophyll a was also found in this complex. It was shown that the molecular ratios of chlorophylla, cis and all-trans-α-carotene to Cyt f in this complex were 1.2, 0.7 and 0.2, respectively.     

Species specific chlorophyll a fluorescence-temperature profile at high temperatures in the leaves

Chlorophyll a (Chl a) fluorescence-temperature profile in the region of 20–80°C was recorded for fourteen different plant species. In all the species studied, there was a rise in the fluorescence intensity in the region of 45–50°C and around 55°C the fluorescence intensity started to decline. In four of the species (Acacia melanoxylon, Ervatamia montana, Eucalyptus tertecornius and Azardicta indica) tested, there was a secondary rise in the fluorescence intensity around 65–70°C whereas in all other species a sharp decline in the fluorescence intensity was observed at this point. These changes in the fluorescence intensity at high temperatures (65–70°C) appear to be species specific and cannot be explained either in terms of changes in the stoichiometry between the two photosystems or in terms of Chl a fluorescence emission from photosystem I (PS I) at higher temperatures. This conclusion is supported by following observations: (1) there was no definite correlation between the Chl a/Chl b ratio and the pattern of fluorescence-temperature profile at high temperatures; (2) the sun and shade plants of the same species had a similar pattern of fluorescence-temperature profile; and (3) preferential excitation of PS I did not alter the fluorescence-temperature profile.Abbreviations Chl chlorophyll - PS photosystem     

Effect of 6-benzyladenine and Casein Hydrolysate on Micropropagation of Amorpha fruticosa

Pisum sativum L. cv. Phenomen plants were grown in pots in greenhouse and their growth, and ATP and chlorophyll (Chl) a and b contents were assessed after 9-d exposure to sodium arsenate [0.04 and 0.07 mmol kg^–1(soil)], or to lead acetate [2.0 and 4.0 mmol kg^–1(soil)], or zinc acetate [5.3 and 9.3 mmol kg^–1(soil)]. The luciferin-luciferase method was used for ATP analyses. Soil pollution reduced significantly the growth, but the low toxicant concentrations elevated the cotyledon and shoot ATP concentrations per fresh matter content. The ATP/Chl ratio was increased in the zinc-treated seedlings as compared with the respective controls. The ATP concentration and a number of growth parameters were negatively correlated, and thus the high ATP content might contribute to the significantly reduced growth of seedlings.     

Nitrogen fixation associated with a hotspring green alga

A unicellular alga which can grow in the light without a combined nitrogen source was isolated from a hot spring. The cells were almost spherical, usually 5–10 m in diameter. Absorption spectra of the watersoluble pigments and of the acetone-extracted ones revealed the existence of chlorophyll a and b and the absence of phycobilins. Thin sections examined by electron microscopy revealed an eukaryotic organization with features typical of the coccoid green algae (the Chlorococcales). Cells divided by internal cytokinesis and subsequent liberation of daughter cells from the parental wall, in a way similar to Chlorella. The alga reduced acetylene to ethylene and incorporated ¹⁵N₂ into cell protoplasm when incubated in a low oxygen atmosphere. Nitrogenase activity was light-dependent, microaerophilic and thermophilic. Although the association of symbiotic nitrogen fixing prokaryotes with the cells may still be possible, any such organisms have not so far been detected.Abbreviations Used DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - Chl chlorophyll - MBM modified Bristol medium - TLC thin layer chromatography     

Seasonal reversibility of acclimation to irradiance in leaves of common box (Buxus sempervirens L.) in a deciduous forest

Understorey shade plants are seasonally exposed to dramatic changes in light conditions in deciduous forests related with the dynamics of the overstorey leaf phenology. These transitions are commonly followed by changes in herb plant communities, but shade-tolerant evergreen species must be able to adapt to changing light conditions. In this work we checked the photoprotective responses of evergreen species to acclimate to the shady summer environment and reversibly de-acclimate to a more illuminated environment after leaf fall on deciduous overstoreys. For that purpose we have followed the process of light acclimation in leaves of common box (Buxus sempervirens) during the winter to spring transition, which decrease irradiance in the understorey, and conversely during the transition from summer to autumn. Four parameters indicative of the structure and degree of acclimation of the photosynthetic apparatus have been studied: chlorophyll a/b ratio which is supposed to be inversely proportional to the antenna size, α/β-carotene which increases in shade acclimated leaves and the pools of α-tocopherol and xanthophyll cycle pigments (VAZ) which are two of the main photoprotection mechanisms in plants. Among these parameters, chlorophyll a/b ratio and VAZ pool responded finely to changes in irradiance indicating that modifications in the light harvesting size and photoprotective capacity contribute to the continuous acclimation and de-acclimation of long-lived evergreen leaves.     

Changes of the concentrations of chlorophyll and phaeopigment in grazing experiments

In grazing experiments, the changes in fluorometric determined concentrations of chlorophyll a and phaeopigment are probably less bothered by the interference of other chlorophyll pigments than in analysis of single seawater samples. Theoretically, phaeopigment recovery will be ca 100%, regardless the eventual presence of chlorophyllb. In shipboard experiments in the northern North Sea in May 1983 the chlorophyll consumption by zooplankton was compared with phaeophorbide egestion. Phaeophorbide recovery amounted on average to 67%, comparable with a previous estimate in laboratory experiments by SHUMAN & LORENZEN (1975). Because phaeophorbide concentrations are expressed in chlorophyll equivalents, one third of the chlorophyll consumed is apparently degraded further than phaeophorbide into non-fluorescent material. In grazing experiments withCalanus finmarchicus stages CIV, CV and CVI filtering rates were determined both with the chlorophyll method and with a particle counter. Values roughly doubled with stage and averages per stage did not differ systematically between both methods, suggesting that the natural algae food, dominated by the microflagellateCorymbellus aureus, was not preferred above other seston.     

Chlorophyll-a determination with ethanol – a critical test

Chlorophyll-a content is widely used as an indicator of the quality of freshwater bodies. Quantification of chlorophyll-a is a routine procedure in the test laboratories of water works, and in research laboratories. Although attempts have been made to standardise the measurement procedure, there are nonetheless many procedures currently in use. This work is focused on a careful re-examination of the ISO: 10260, 1992 standard, which prescribes 90% (v/v) ethanol for chlorophyll extraction and measurement. Chlorophyll contents of cultures of the cyanobacterium Synechococcus elongatus Nägeli and the chlorophyte Scenedesmus acutus Meyen were determined by means of a series of concentrations of ethanol/water mixtures which were employed as extracting agents – the water content was gradually decreased from 20 to 0%. The extraction procedure was verified by measuring the amount of retained water after using both water and oil pumps for filtering the samples. The spectroscopic effects of the presence of water were studied and the molecular background of these spectral phenomena is discussed. The extraction yields obtained with 90% ethanol were compared to those obtained with methanol and acetone. On the basis of the calculated error level, improvements to the ISO: 10260, 1992 standard method have been suggested.     

Factors affecting the accuracy of chlorophylla andb determination by means of their paper chromatographic separation and colorimetric measurement in eluates

Quantitative determination of chlorophyll a and β can be made by paper chromatography of acetone extracts of plant material with colorimetric measurement of the eluates from the separated zones. From the suitable solvent systems which give adequate separation of the pigments at a distance of 20 cm. from the start,Hager's mixture (1955) separates the chlorophylls better than the toluene-isopropanol (400: 1 v/v.) mixture, which, however, is better for the separation of carotenoids. Twice the amount of chlorophyll is separated on Whatman 31 ET paper, equally well and with the same time of development, as on Whatman No. 3 paper, on which it is possible to separate a maximum of about 15 μg of chlorophyll pigments per 1 em. start length. Losses on elution are, however, higher on using Whatman 31 ET paper. In plants with a high chlorophyllase activity, the error of determining chlorophyll a andb is greatly reduced if the leaves are placed for 1 min. in boiling water before extraction. For elution of chlorophylla andb from paper it is better to use anhydrous acetone, for chlorophyllides 80% acetone. A comparison of the procedure investigated with the method of two-wave length spectrophotometric measurement of crude acetone extracts showed that in view of the average 10% loss, the chromatographic method is hardly suitable for determining the absolute amounts of chlorophylla andb, although the relation (a/b) can be determined with similar precision by both methods. Moreover, in view of the greater amount of work involved the chromatographic method can only be recommended for confirming the results of spectrophotometrie determination. Quantitative determination of chlorophylls from the area of the spot or from the "R_F" value can only be of an informative character.     

Spatial patterns of benthic diatoms,carbohydrates and mud on a tidal flat in the Ems-Dollard estuary

The chlorophyll a content and two operational fractions of carbohydrate (water extractable and EDTA extractable) were measured every three months during one year along transects on a tidal flat in the Ems-Dollard estuary (The Netherlands). Chlorophyll a was used as an indicator of microphytobenthos biomass, which was composed predominantly of epipelic diatoms. Both carbohydrate fractions correlated significantly with chlorophyll a. EDTA extractable carbohydrates were more resistant towards degradation than the water extractable fraction. During most of the year, concentrations of chlorophyll a and carbohydrates were low, but in June, high concentrations of up to 90 g chlorophyll a/g sediment were found in a narrow zone running parallel to the channel. Maximum concentrations of water extractable carbohydrates and EDTA extractable carbohydrates ranged between 800–1200 and 600–800 g/g sediment, respectively. The mud content was high (± 90%) at the margin of the tidal flat. This was not limited to the growth season of the diatoms, but was observed throughout the year. This indicated that the high mud content at the mudflat margin was mainly caused by hydrodynamic factors, and not by biostabilization. In June, exceptionally high diatom densities were found in sediment with a high mud content. There was only minor evidence that biostabilization by epipelic diatoms lead to a further increase in the mud content of the sediment.     

Predicting concentration of total phosphorus and chlorophyll a in a lake with short hydraulic residence time

The relationship between total phosphorus and chlorophyll a concentration was determined for Skinner Lake, Indiana over an annual cycle in 1978–79. Total nitrogen:total phosphorus ratios in the epilimnion ranged from 19 to 220 suggesting a phosphorus-dependent algal yield in the epilimnion. Approximately 90% of annual TP loading reached the lake via streamflow, and 93% of this entered during snowmelt and spring-overturn periods. At that time incoming water flushed the lake 2.4 times. Atmospheric loading accounted for 1.4% of annual TP load. Internal hypolimnetic TP loading occurred during summer stratification. Mean [chl a] for the ice-free period was 15.15 mg m^–3, within the range expected for eutrophic lakes.The 1978–79 data were used in conjuction with the Vollenweider & Kerekes (1980) model to produce a model specific for the Skinner Lake system. The model predicted mean epilimnetic total phosphorus and chlorophyll a concentrations from mean total phosphorus concentration in inlet streams and from lake water residence time during the period of spring overturn and summer stratification. The Skinner-specific model was tested in 1982 and it closely predicted observed mean epilimnetic [TP] and [chl a] during the ice-free period. This study shows that variability in lake models which average data over an annual period can be reduced by considering lake-specific seasonal variation in hydrology and external TP loading.     

The effect of non-algal turbidity on the relationship of Secchi depth to chlorophyll a

Data from four reservoirs representative of different trophic states and with different apparent optical properties were analyzed to determine the relationship of Secchi depth to algal biomass as measured by chlorophyll a. In the eutrophic reservoir Secchi depth was determined partially by the chlorophyll a content (r² = 0.31) but only when chlorophyll a data from bloom conditions are included. In the two mesotrophic reservoirs, Secchi depth was entirely determined by non-algal turbidity. In the oligotrophic reservoir, Secchi depth was determined neither by chlorophyll a nor non-algal turbidity and was probably determined by dissolved color. When data from the four reservoirs were pooled (N = 205), 53% of the variation in Secchi depth was explained by: SD = 2.55–0.52 ln (Turbidity) + 0.005 (Chlorophyll a). It is apparent that attempts to estimate algal biomass for trophic state classification or other management practices from Secchi depth data are inappropriate even where moderate amounts of non-algal turbidity are present.