Analysis of the chlorophyll biosynthetic system in a chlorophyll b-less barley mutant

The chlorophyll b-less barley (Hordeum vulgare L.) mutant chlorina 2807 allelic to the well-known barley mutant chlorina f2 was studied. 5-Aminolevulinic acid at saturating concentration (40 mM) was introduced into postetiolated leaves of the mutant and its wild type, and the protochlorophyllide accumulation in the dark was measured. It was found that the activity of the enzyme system transforming 5-aminolevulinic acid into protochlorophyllide was the same in both types of plants. The activity of esterifying enzymes that catalyze attachment of phytol to chlorophyllide was analyzed by infiltration of exogenous chlorophyllides a and b into etiolated leaves. The reaction was shown to have close rates in the mutant and wild-type plants. In very early stages of greening of etiolated leaves, when the apoproteins of the light-harvesting complexes are not yet formed, appearance of chlorophyll b was clearly recorded in the wild-type plants, while in the mutant chlorina 2807 no indications of chlorophyll b were detected in any stage of greening. On the other hand, in the mutant as well as in the wild type an active reverse conversion of chlorophyll b into chlorophyll a was possible. It is concluded that (a) in the mutant chlorina 2807 the ability of the biosynthetic system to transform 5-aminolevulinic acid to chlorophyll a is fully preserved, (b) in the mutant the enzymes converting chlorophyll a into chlorophyll b are most likely absent or damaged, (c) the conversion of chlorophyll a into chlorophyll b and the reverse conversion of chlorophyll b into chlorophyll a are performed by different enzymes.     

Simultaneous estimation of chlorophyll a and lipid contents in microalgae by three-color analysis

A method of rapid determination of chlorophyll a and lipid contents of microalgae based on colorimetric analysis of the digital images of the microalgae is proposed. The color variation of microalgae during cultivation is evaluated by the brightness of the three primary colors (red, green, and blue). The brightness values of the three primary colors are modeled as two linear correlation functions (RGB model) for microalgal chlorophyll a and lipid contents, respectively. The chlorophyll a and lipid contents predicted by the proposed model are compared with that determined by the standard methods. The good agreement of the model predictions with experimental results is demonstrated with a squared correlation coefficient (R(2)) of 0.99 for chlorophyll a and lipid. The reliability of the RGB model was verified in real cultivations of the microalgae in a photobioreactor. Growth dynamics, contents of chlorophyll a and lipid corresponded very well with previously reported studies.     

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.     

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.     

A chemotaxonomic method to quantify phytoplankton groups in freshwater lentic mesocosms: an approach including chlorophyll a breakdown products

The use of HPLC methods to determine and quantify phytoplankton population composition, is sometimes less time-consuming than microscopic identification. However, its general application poses problems since high discrepancies between chlorophyll a calculated using chemotaxonomic methods and direct measurements were noticed. For instance, chemotaxonomic protocols generally employed can lead to a poor estimation of total and relative abundance when high amounts of chlorophyll a breakdown products are present. Therefore, we propose a new approach to calculate relative abundance of algal groups in a phytoplankton population, based on integration of these degradation products in the chemotaxonomic assessment in lentic and shallow freshwater ecosystems.     

A liquid chromatography–mass spectrometry platform for the analysis of phyllobilins,the major degradation products of chlorophyll in Arabidopsis thaliana

During senescence, chlorophyll is broken down to a set of structurally similar, but distinct linear tetrapyrrolic compounds termed phyllobilins. Structure identification of phyllobilins from over a dozen plant species revealed that modifications at different peripheral positions may cause complex phyllobilin composition in a given species. For example, in Arabidopsis thaliana wild‐type, eight different phyllobilins have structurally been characterized to date. Accurate phyllobilin identification and quantification, which classically have been performed by high performance liquid chromatography (HPLC) and UV/vis detection, are, however, hampered because of their similar physiochemical properties and vastly differing abundances in plant extracts. Here we established a rapid method for phyllobilin identification and quantification that couples ultra‐HPLC with high‐resolution/high‐precision tandem mass spectrometry. Using Arabidopsis wild‐type and mutant lines that are deficient in specific phyllobilin‐modifying reactions, we identified a total of 16 phyllobilins, among them two that have not been described before in Arabidopsis. The single and collision‐induced dissociation tandem mass spectrometry data of all 16 Arabidopsis phyllobilins were collected in a mass spectrometry library, which is available to the scientific community. The library allows rapid detection and quantification of phyllobilins within and across Arabidopsis genotypes and we demonstrate its potential use for high‐throughput approaches and genome‐wide association studies in chlorophyll breakdown. By extending the library with phyllobilin data from other plant species in the future, we aim providing a tool for chlorophyll metabolite analysis as a measure of senescence for practical applications, such as post‐harvest quality control.     

Benthic microalgae: comparisons of chlorophyll a in mesocosms and field sites

A mesocosm facility is being developed by the CSIRO Division of Fisheries to study the movement, fate and impact of pollutants in coastal marine environments. Initially, we are studying coastal habitats in S.W Australia that are subject to wave action. A 12 week experiment assessed changes in biotic and abiotic components of sediment brought from the field into mesocosms. In these sediments, benthic microalgae are the major primary producers. Microalgal standing stocks were measured as the concentration of chlorophyll a and phaeopigments in the top 2 cm of sediment, and these measures were compared among mesocosms and field sites. Significant increases in chlorophyll a and phaeopigments, increased numbers of species and a shift from episammic to periphytic diatoms were observed, possibly caused by decreased water motion in mesocosms or other containment effects. Results from statistical power analyses suggested that our sampling was sufficiently well replicated and successfully incorporated variation at small spatial and temporal scales. Sampling effort used in this experiment should form the basis for future work with benthic microalgae.     

Characteristics of nutrient dynamics in Lake Sagate,Japan, a shallow sand dune lake

We examined differences in digestibility and viability following gut passage through water penny larvae (Psephenus herricki) of Synedra ulna and Achnanthidium lanceoloatum, two common diatom taxa that differ in growth habit and autecological characteristics. Prior to the experiment, diatoms were cultured in Chu-10 media in petri plates to establish a monospecific biofilm to offer grazers. After collection, insects were left to clear their guts over night, allowed to graze for 3 hours on diatom biofilms, and then placed in vials over 1-mm mesh to defecate. Samples from source material and from insect feces were mounted in syrup media and the ratio of chloroplast-containing to empty diatom frustules was microscopically assessed. In addition, subsamples from source material and feces were sprayed onto agar plates prepared with Chu-10 and individual cells were mapped and tracked for 5 days to quantify reproduction. Cells of both S. ulna and A. lanceolatum taken from source material formed colonies on agar. Achnanthidium lanceolatum cells from insect feces also formed colonies, but with lower densities than those from source material. In contrast, none of the S. ulna cells tracked from fecal cultures formed colonies, and the percentage of S. ulna cells that were dead was significantly greater in feces relative to source material. Dead cell percentages of A. lanceolatum were also higher in feces relative to source material, but to a lesser degree than observed for S. ulna. These findings have potential implications for linking patterns of energy transfer in stream ecosystems and the structure and dynamics of benthic microalgal communities.     

Gradient of dissolved free amino acids and phytoplankton in a shallow bay

A 3 h survey of the concentrations of individual free amino acids, chlorophyll a and phytoplankton species biomass was conducted in the surface waters of a shallow bay. Significant coefficients of correlation were found between chlorophyll a, nanoflagellates, and DFAA concentrations. Although phytoplankton biomass variations sometimes relate to DFAA concentration patterns, consideration of the physiological activity of both phytoplankton and microheterotrophs would undoubtedly explain a more significant fraction of the DFAA variation.This work is a contribution of GREPMA (Groupe Régional d'Etudes Pélagiques en Manche-Atlantique)     

Plankton ecology in an ice-covered bay of Lake Michigan: utilization of a winter phytoplankton bloom by reproducing copepods

Plankton ecology was examined during the 1986 winter in Grand Traverse Bay, a 190 m deep, fjordlike bay on Lake Michigan. Before ice cover, algal concentration was low and uniformly distributed with depth, as it is in open Lake Michigan. During ice cover (February and March), a bloom of a typical winter-spring phytoplankton community developed in the upper 40 m, resulting in a 4 to 7-fold increase in feeding rate of adult Diaptomus spp. High algal concentration and zooplankton feeding persisted after ice melt (April). During and after ice cover, lipid concentrations of Diaptomus dropped rapidly from 34% of dry weight to 17 % because of egg production. High incident photosynthetically active radiation (PAR), high (45–50%) PAR transmittance of the ice due to little snow on the ice, and water column stability were probably responsible for the bloom. High ice transparency may be a common feature of large lakes and bays, where strong winds blow snow cover off the ice, or at low latitudes where snowmelt due to occasional rains and warm temperature is common. Winter reproducing calanoid copepods use these blooms to increase their reproductive output.     

Artificial leaf aids analysis of chlorophyll fluorescence and P700 absorbance in studies involving microalgae

Measuring chlorophyll fluorescence and P700 absorbance has been widely used to study photosynthesis in both terrestrial plants and algae. However, in order to apply these measurement techniques to study microalgae, a concentrated suspension of algae, which is usually prepared by centrifugation, is required. In this study, instead of using centrifugation, we concentrated microalgae on a nitrocellulose membrane using filtration to create an ‘artificial leaf’ before analysis. Overall, we were able to generate values of the appropriate photosynthetic parameters that were comparable to those obtained when chlorophyll fluorescence and P700 absorbance were measured following centrifugation. There were no statistically significant differences (P > 0.05) between the artificial leaf method and the traditional cuvette method for determining chlorophyll fluorescence or P700 absorbance at appropriate chlorophyll concentrations. We were also able to reduce background noise by using a filter membrane as a carrier. Therefore, an artificial leaf has the potential to be a valuable tool for phycologists interested in studying microalgal photosynthesis by enabling them to eliminate tedious centrifugation steps. In addition, fluorometers commonly used for studying the leaves of higher plants will also be suitable for studying microalgae.     

Characterization of potato genotypes by chlorophyll fluorescence during plant aging in a Mediterranean environment

Field experiments were conducted in Sicily (south Italy) during two seasons to characterize by chlorophyll (Chl) fluorescence four genotypes (Spunta, Sieglinde, Daytona, and Ninfa) of potato (Solanum tuberosum L.) for off-season production during plant aging and to analyse the possible relation between Chl parameters and tuber yield. Chl fluorescence parameters [initial fluorescence (F₀), maximum fluorescence (F_m), F_v/F_m, time in which maximal fluorescence occurs (T_max)] gained from Kautsky kinetics and Chl content were measured weekly, from 5^th to 6^th leaf appearance to beginning of plant senescence in the first season and to full plant senescence in the second season. F₀ and F_v/F_m were the most reliable Chl fluorescence parameters for the definition of genotypic differences while Chl content and T_max were the most reliable Chl parameters to predict plant aging. Tuber yield was highly correlated with Chl content, T_max, F₀, and F_m.     

Responses of benthic algae to nutrient enrichment in a shallow lake: Linking community production,biomass, and composition

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Small-scale distribution of juvenile plaice and flounder in relation to predatory shrimp in a shallow Swedish bay

Metamorphosed plaice and flounder were sampled in a small non-tidal nursery bay with a non-selective drop trap from April to August in 1991 and 1992. Post-settlement patterns were mapped in time and space and related to wind stress and the distribution of a potential predator Crangon crangon L. Both plaice and flounder had a patchy distribution at a sample scale of 1 m². Plaice settlement coincided with timing of onshore winds in 1991 and 1992, while flounder settlement seemed to be unrelated. Plaice settled earlier than flounder and during the early phase of 1-group C. crangon immigration. The first appearance of flounder occurred simultaneously with the peak biomass of predatory C. crangon . Plaice occurred in the deeper part of the bay together with C. crangon , while flounder preferred shallow shore waters. We propose that different temporal (plaice) and spatial (flounder) strategies have evolved in order to minimize the risk of shrimp predation after settlement.     

Palaeolimnological aspects of a Late-Glacial shallow lake in Sandy Flanders,Belgium

A summary account is given of the development of a small Late-Glacial lake at Snellegem-St. Andries, Belgium. Sedimentation, hydrology, water quality and biotic succession clearly depended on climatic conditions and catchment processes (soil stability and leaching, vegetation). Special attention is drawn to a period of low water level near the end of the Allerød and the abundance of Fragilaria in certain periods.     

Ingestion and digestion of epilithic algae by larval insects in a heavily grazed montane stream

1. Epilithic algae grown on elevated or non-elevated ceramic tiles were exposed (to produce assemblages with different grazing histories) in a heavily grazed, montane stream in New Mexico, U.S.A. to Ameletus nymphs (Ephemeroptera) and Ecclisomyia larvae (Trichoptera) and the algal composition in insect faeces was compared to that on the tiles. Differences in grazing and digestion efficiency between grazers were then assessed and also differences in susceptibility to ingestion and digestibility among common algae. 2. Ordination of tile and faecal samples, using the relative abundance of common algae, revealed that: (i) algal assemblages on elevated vs. non-elevated tiles differed only slightly; (ii) the taxonomic composition of algae in faeces of both caddis and mayflies differed substantially from that on the tiles, indicating low grazing efficiency for some algal taxa; and (iii) the algal composition of faeces produced by caddis larvae and mayflies was similar, indicating little difference in grazing efficiency between them. However, some algal taxa were more susceptible to ingestion by caddisfly larvae when occurring on elevated tiles than on non-elevated tiles, suggesting that previous exposure to caddis grazing influenced assemblage attributes. 3. Although Ameletus and Ecclisomyia differed little in grazing efficiency, the percentage of diatoms that were dead after passage through the gut was greatest in the mayfly treatment, suggesting that mayflies digested diatoms more efficiently than the caddis. Analyses of differences in the condition of chloroplasts within diatoms in tile and faecal samples showed that losses of ’live‘ diatom cells (i.e. those containing full chloroplasts) during gut passage through mayflies equalled the increase, in faeces, of ’dead‘ (empty frustules) cells of all common diatoms. In contrast, some diatoms were digested inefficiently by caddis larvae. 4. Algae on elevated tiles contained a higher proportion of dead diatoms than those on non-elevated tiles, possibly because mayflies visited raised tiles more often and, consequently, ingested and defaecated cells at a higher rate in the absence of caddis larvae. Moreover, diatom taxa differed in the percentage of cells that were dead within tile assemblages, with populations of typically grazer-resistant taxa (e.g. Achnanthidium minutissimum, Planothidium lanceolatum and Cocconeis placentula var. euglypta) containing significantly more dead cells than grazer-susceptible taxa [e.g. small, chain-forming Fragilaria (= Staurosirella)]. This result suggests that a trade-off exists between ingestion vs. digestion resistance of microalgae. Both the ingestion and digestion efficiency of algivorous macroinvertebrates could influence the structure and function of algal assemblages. In heavily grazed systems, where algal cells are probably processed through grazer guts repeatedly, differential resistance to digestion among algae may be particularly important.     

The distribution of NADPH-protochlorophyllide oxidoreductase in relation to chlorophyll accumulation along the barley leaf gradient

The possible regulatory role of NADPH-protochlorophyllide oxidoreductase for chlorophyll accumulation has been investigated in barley plants. Within the primary leaf of etiolated plants the different maturation stages of etioplasts are found in a linear series with the youngest in cells near the base and the oldest in cells near the tip. This distribution of different plastid forms is paralleled by drastic differences in the NADPH-protochlorophyllide-oxidoreductase content of the plastids and their capacity to accumulate chlorophyll during illumination. The amount of enzyme and the rate of chlorophyll accumulation are highest in the mature etioplast in the tip of the leaf and both decline rapidly with decreasing age of the leaf tissue, being almost undetectable in the leaf base. The translatable mRNA coding for the enzyme shows a different distribution pattern within the leaf. The highest concentration is found in the middle part of the leaf while in the top part only traces of this mRNA are detectable. It is concluded that during leaf development the enzyme is synthesized rapidly only during a limited time period and that it is stored subsequently in the mature etioplast as a stable protein. The close correlation between the distribution of the enzyme within the barley leaf and that of the potential to accumulate chlorophyll during illumination would favour a control of chlorophyll accumulation by the amount of NADPH-protochlorophyllide oxidoreductase. Dark-grown plants which were exposed to far-red light were used to test this possibility. The far-red-absorbing form of phytochrome (P_fr) has an inverse effect on the kinetics of chlorophyll accumulation and the enzyme concentration. Our results indicate that the rate of chlorophyll accumulation in barley is not determined by the level of NADPH-protochlorophyllide oxidoreductase present in the leaves.     

HPLC analysis of phytoplankton pigments from Lake Kinneret with special reference to the bloom-forming dinoflagellate Peridinium gatunense (Dinophyceae) and chlorophyll degradatio products

Photosynthetic pigments extracted from the paniculate materialof the water column of Lake Kinneret were studied throughoutthe periods of May 1988-June 1989, and November 1993-November1994, by means of HPLC. The temporal and vertical variationof the pigment suite found agreed with the microscopically determinedphytoplankton record. The regression calculations of taxon-specificbiomass with the corresponding signature pigments suggest thatpigment analysis may be a useful tool for the monitoring ofbloom-forming species, e.g. the dinoflagellate Peridinium gatunenseNygaard. The HPLC pigment analysis permitted the identificationand quantification of chlorophyll degradation products, providingfor the first time information about their composition in LakeKinneret. Chlorophyllide a was the major detectable degradationproduct of chlorophyll a, varying between 1 and 9% of the chlorophylla concentration. Other chlorophyll a derivatives appeared mostlyin minor quantities. Pheophytin a was virtually lacking in allthe samples. Removal rates of pigments, measured by sedimentationtraps, indicated that the degradation of chlorophyll a via chlorophyllidea is a dynamic process that continues during the sedimentationof the phytoplankton particles.