Algal Chloroplasts Research Articles

Dynamics of a peculiar plant-herbivore relationship: the photosynthetic ascoglossan Elysia timida and the chlorophycean Acetabularia acetabulum

The ascoglossan mollusc Elysia timida Risso, 1818 retains functional chloroplasts from its algal food, the chlorophycean Acetabularia acetabulum (L.). Photosynthates from the plastids are an important source of organic nutrients for the mollusc. Chloroplast exploitation has an ecological function, allowing the ascoglossan to live entirely on an algal diet which is of limited, seasonal availability to other herbivores. Between October 1987 and July 1988, the annual evolution of the molluscan and algal populations was studied in a cove of Mazarron Bay, southeast Spain. The population density of the mollusc is highly dependent on its food supply, being controlled by the seasonal life cycle of the algal population. During its life cycle, the degree of grazing by the mollusc decreases with increasing algal calcification, the cell walls of the alga progressively calcify, and the eventually highly calcificied stalks are completely resistant to ascoglossan grazing. In contrast, the exploitation of the algal chloroplasts retained by the molluscs increases during the seasonal cycle. The progressively increasing scarcity of food during the seasonal cycle may have led to the retention of symbiotic chloroplasts by E. timida. The developmental strategy of the ascoglossan also changes during the year: when food is abundant (in November, December, January, February and March) it is direct, with no planktonic larval phase, when food is scarce (in October, April, May and June) it is lecithotrophic, with a short planktonic larval phase. Chloroplast retention acts as a buffer, alleviating the effects of annual changes in density, structure and abundance of the alga on the nutritional state of the molluse.

Structural organization of the chloroplast genome of the chromophytic algaVaucheria bursata

The chloroplast genome of the chromophytic alga Vaucheria bursata has been characterized by restriction site and gene mapping analysis. It is represented by a circular molecule 124.6 kb in size. An inverted sequence duplication (IR) not larger than 5.85 kb carries the rRNA genes and separates two single-copy regions of 64.6 kb and 48.3 kb from one another. The Vaucheria plastid genome exists in two equimolar isomers which is due to intramolecular flip-flop recombination within the IR sequences. The coding sites for 21 structural and soluble proteins have been mapped on both single-copy regions using heterologous gene sequences as probes. Although the overall gene order is found to be rearranged when compared with other chromophytic algal and land plant chloroplast genomes, most of the transcriptional units of cyanobacteria and land plant chloroplast genomes appear to be conserved. The phylogenetic implications of these findings are further discussed.

The Supramolecular Organization of Photosynthetic Membranes of the Thallus Stage of Porphyra leucosticta Thuret. (Bangiales, Rhodophyta) Visualized by Freeze‐Fracture

AbstractThe supramolecular organization of the thylakoid membranes of the thallus stage in the red alga Porphyra leucosticta is studied in replicas of rapidly frozen and fractured cells. Freeze‐fractured thylakoid membranes exhibit only two types of fracture faces (EF and PF), because the lamellae in red algal chloroplasts are not stacked. The PF reveals numerous, tightly packed, but randomly distributed particles (density range from 2970 to 3550 particles/μm2). In contrast, the EF particles appear organized into parallel rows, the spacing of which is about 60–70 nm (about 8–9 particles occur along 100 nm of the line that is formed). Significant numbers of single EF particles are randomly distributed between the EF particle rows. The particles on both fracture faces (PF and EF) fall into two size classes: 10 to 11 nm (major size class) and 14 to 15 nm (minor size class).

Localization of Nitrogen-Assimilating Enzymes in the Chloroplast of Chlamydomonas reinhardtii

The specific activities of nitrate reductase, nitrite reductase, glutamine synthetase, glutamate synthase, and glutamate dehydrogenase were determined in intact protoplasts and intact chloroplasts from Chlamydomonas reinhardtii. After correction for contamination, the data were used to calculate the portion of each enzyme in the algal chloroplast. The chloroplast of C. reinhardtii contained all enzyme activities for nitrogen assimilation, except nitrate reductase, which could not be detected in this organelle. Glutamate synthase (NADH- and ferredoxin-dependent) and glutamate dehydrogenase were located exclusively in the chloroplast, while for nitrite reductase and glutamine synthetase an extraplastidic activity of about 20 and 60%, respectively, was measured. Cells grown on ammonium, instead of nitrate as nitrogen source, had a higher total cellular activity of the NADH-dependent glutamate synthase (+95%) and glutamate dehydrogenase (+33%) but less activity of glutamine synthetase (-10%). No activity of nitrate reductase could be detected in ammonium-grown cells. The distribution of nitrogen-assimilating enzymes among the chloroplast and the rest of the cell did not differ significantly between nitrate-grown and ammonium-grown cells. Only the plastidic portion of the glutamine synthetase increased to about 80% in cells grown on ammonium (compared to about 40% in cells grown on nitrate).

Chloroplast envelope proteins from Chlamydomonas: separation into outer and inner envelopes and analysis by two-dimensional gel electrophoresis

Fractions enriched in outer and inner chloroplast envelope membranes were isolated from the alga Chlamydomonas reinhardtii. Functionally active chloroplasts were lysed after equilibration in a hypertonic solution and outer and inner envelopes were separated. The protein content of each fraction was analysed by two-dimensional gel electrophoresis using isoelectric focusing/SDS polyacrylamide gel electrophoresis. The method is capable of separating components over a wide range of molecular mass and p I without interference by lipid binding. Each fraction contained a characteristic protein pattern. A 32 kDa protein was found exclusively associated with the inner envelope indicating that algal chloroplast envelopes share a common feature with those of higher plants.

Characterization of the pyrenoid isolated from unicellular green algaChlamydomonas reinhardtii: Particulate form of RuBisCO protein

The pyrenoid is a protein complex in the chloroplast stroma of eukaryotic algae. After the treatment with mercury chloride, pyrenoids were isolated by sucrose density gradient centrifugation from cell-wall less mutant cells, CW-15, as well as wild type cells, C-9, of unicellular green algaChlamydomonas reinhardtii. Pyrenoids were characterized as a fraction whose protein/chlorophyll ratio was very high, and also examined by Nomarski differential interference microscopy. Most of the components consisted of 55 kDa and 16 kDa polypeptides (1∶1) which were immunologically identified as the large and small subunit of RuBisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase) protein, respectively. Some minor polypeptides were also detected. Substantial amount of RuBisCO protein is present as a particulate form in the pyrenoid in addition to the soluble form in algal chloroplast stroma.

31] Phycobiliproteins

This chapter focuses on Phycobiliproteins. In intact cyanobacterial cells and red algal chloroplasts, the phycobiliproteins are components of a complex assemblage, the phycobilisome. The observation that upon cell breakage the products of phycobilisome dissociation are released in water-soluble form gave rise to the unfortunate impression that the phycobiliproteins represented a fairly simple family of well-behaved macromolecules. Appraisal of the true complexity of the situation required an understanding of the molecular structure of phycobilisomes. During purification procedures, certain phycobilisome polypeptide components are lost through retention on chromatographic supports, or, in some cases, destroyed by proteolysis. Some phycobiliproteins, such as allophycocyanin B, are products of subunit scrambling during purification procedures, while others, such as the β18.3 polypeptide of the phycobilisome core, which is present in only two copies per phycobilisome, may copurify undetected with one of the major phycobiliproteins such as allophycocyanin. A polypeptide pattern obtained by SDS-polyacrylamide gel electrophoresis is an absolutely essential element in the characterization of a purified phycobiliprotein preparation. A sufficient amount of protein should be applied to the gel to ensure that components present in small amounts are detected.

Immunocytochemical Localization of Phosphoribulose Kinase in the Cyanelles of Cyanophora paradoxa and Glaucocystis nostochinearum

The distribution of phosphoribulose kinase (PRK) in the cyanelles of Cyanophora paradoxa Korschikoff and Glaucocystis nostochinearum Itzigsohn was studied by protein A-gold immunoelectron microscopy. In both endocyanomes, antiserum against PRK heavily labeled the thylakoid region of the cyanelles, whereas little or no label was present over the carboxysomes. Antiserum against ribulose 1,5-bisphosphate carboxylase/oxygenase by contrast heavily labeled the carboxysomes of each endocyanome. In vitro studies of PRK distribution in cell-free extracts of C. paradoxa showed that 93% of the enzyme was in the soluble fraction. Quantitative immunoelectron microscopy showed that more than 99% of the PRK in the cyanelle of C. paradoxa was localized in the thylakoid region. We conclude that the carboxysomes of cyanelles like the carboxysomes of autotrophic prokaryotes and the pyrenoids of green algal chloroplasts do not contain phosphoribulose kinase.

Ultrastructure and ontogeny of hair filaments in the subtidal saccate alga Hydroclathrus clathratus (Phaeophyta, Scytosiphonales)

Hair filaments found in pits scattered over the surface of the saccate brown alga Hydroclathrus clathratus were examined ultrastructurally. Cells near the base of filaments are short and metabolically very active, containing many mitochondria, ribosomes, rough endoplasmic reticulum, and dictyosomes but no vacuoles. Numerous plasmodesmata connect cells of the filaments. The more distal cells elongate during development of the pits. As elongation begins, dictyosome activity dominates and vacuoles appear. Very elongate cells are filled almost exclusively by a vacuole with a narrow band of cytoplasm at the cell periphery. Chloroplasts are found only in basal cells of short filaments. Other cells near the filament base contain plastosomes, small spherical bodies derived from chloroplasts by a series of divisions. While morphologically distinct, plastosomes have retained many features of brown algal chloroplasts. They are bound by the chloroplast envelope and chloroplast endoplasmic reticulum and contain a ring genophore. The thylakoid system varies in that more than three thylakoids are usually loosely associated in the peripheral band and the interior bands are absent in more mature plastosomes.

Import of proteins into chloroplasts. Membrane integration of a thylakoid precursor protein reconstituted in chloroplast lysates.

Many of the thylakoid membrane proteins of plant and algal chloroplasts are synthesized in the cytosol as soluble, higher molecular weight precursors. These precursors are post-translationally imported into chloroplasts, incorporated into the thylakoids, and proteolytically processed to mature size. In the present study, the process by which precursors are incorporated into thylakoids was reconstituted in chloroplast lysates using the precursor to the light-harvesting chlorophyll a/b protein (preLHCP) as a model. PreLHCP inserted into thylakoid membranes, but not envelope membranes, if ATP was present in the reaction mixture. Correct integration into the bilayer was verified by previously documented criteria. Integration could also be reconstituted with purified thylakoid membranes if reaction mixtures were supplemented with a soluble extract of chloroplasts. Several other thylakoid precursor proteins in addition to preLHCP, but no stromal precursor proteins, were incorporated into thylakoids under the described assay conditions. These results suggest that the observed in vitro activity represents in vivo events during the biogenesis of thylakoid proteins.

A single chloroplast protein with latent activities of both N ADPH-dependent glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase

An algal chloroplast protein possessing latent NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase and latent phosphoribulokinase activities has been purified. Both activities were stimulated by incubation with dithiothreitol and NADPH. The protein had subunit composition 8G6R and on activation depolymerized to discrete NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase (4G) and phosphoribulokinase (2R). Similar depolymerization promoted by reduced thioredoxin could account for light-dependent activations of phosphoribulokinase and NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase observed in algae and higher plants.

Compartmentation of glycolysis and of the oxidative pentose-phosphate pathway in Chlamydomonas reinhardii

Glycolytic enzyme activities and enzyme activities of the oxidative pentose-phosphate pathway were measured in intact chloroplasts from Chlamydomonas reinhardii. By comparison with the total enzyme activities of intact protoplasts of the alga the data were used to locate these enzymes quantitatively in the algal chloroplast. It was found that the glycolytic chain in C. reinhardii is roughly split into a plastidic and an extraplastidic part. More than 90% of the first part of glycolysis (from fructose-6-phosphate to triose-phosphate is located in the plastid while more than 95% of the second part (from glycerate-3-phosphate to pyruvate) is outside. Around 70% of glucose-6-phosphate dehydrogenase and gluconate-6-phosphate dehydrogenase, two key enzymes of the oxidative pentose phosphate pathway, are located in the plastid. It is concluded that in C. reinhardii the major part of hexose breakdown to triose-phosphate occurs in the chloroplast and that a tight cooperation between the plastid and the cytoplasm is required for appreciable sugar breakdown to occur in the algal cell.

Excitation-energy distribution in green algae. The existence of two independent light-driven control mechanisms

Three distinct states can be identified for cells of the green alga Chlorella vulgaris; State 1 and State 2 obtained by preillumination in far-red and red light, respectively, and the dark state obtained by dark-adaptation. Addition of the inhibitor DCMU to algal cells leads to an initial rapid increase in chlorophyll- a fluorescence reflecting the closure of Photosystem II traps. This, in the case of dark and state-2-adapted algae is followed by a slow light-dependent increase to a fluorescence yield typical of State-1-adapted cells. Measurements of low temperature (77 K) emission spectra indicate that the low fluorescence yields of dark and State-2-adapted algae reflect similar balances in excitation-energy distribution between the two photosystems. In both cases, the balance favours PS I and the slow fluorescence increase seen in the poisoned algae reflects a redressing of this balance in favour of PS II. The low fluorescence yield of State-2-adapted algae is thought to be associated with the phosphorylation of chlorophyll a/b light-harvesting protein (Biochim. Biophys. Acta (1983) 724, 94–103). Measurements of the uncoupler and ATPase sensitivity of the light-dependent increases seen in DCMU-poisoned cells indicate that the low fluorescence yield of dark-adapted algae is of different origin. Evidence is presented showing that the light-driven changes in excitation-energy distribution seen in green algae involve two distinct processes; a low-intensity, wavelenght-independent change reflecting simple light/dark changes and a higher intensity, wavelength-dependent change reflecting State 1/State 2 adaptation. The former changes appear to be associated with changes in the local ionic environment within the algal chloroplast, whilst the latter appear to reflect changes in the phosphorylation state of chlorophyll a/b light-harvesting protein.

Novel Phycoerythrins in Marine Synechococcus spp.

Four clones of the marine, unicellular, cyanobacteria Synechococcus spp., were examined for the spectral and biochemical features of their phycoerythrins (PE) and their photosynthetic characteristics. Two spectral types of PE which are distinct from known PEs were found. One PE type possessed absorption maxima at 500 and 545 nm and a fluorescence emission at 560 nm. Upon denaturation in acid-urea, two chromophore absorption maxima were obtained, one corresponding to phycourobilin (A(max) 500 nm) and one at 558 nm, ascribed to a phycoerythrobilin-like chromophore. The ratio of phycoerythrobilin-like to phycourobilin chromophores was 4.9:1.3. This PE possessed two subunits of M(r)s of 17.0 and 19.5 kD for the alpha and beta subunits, respectively. The other PE possessed a single symmetrical absorption at 551 nm and a fluorescence emission at 570 nm. This phycobiliprotein showed a single chromophore absorption band (A(max) 558 nm) and yielded two polypeptides, an alpha of 17.5 kD and a beta subunit of 20.8 kD. Both PEs showed a (alpha, beta)(n) structure. The presence of phycoerythrobilin-like chromophores (A(max) 558 nm) appears to be diagnostic of this marine cyanobacterial group. The features of these PEs combined with additional biochemical data, suggest a possible evolutionary link between the PE-containing marine Synechococcus group and the red algal chloroplast. When the Synechococcus clones were grown under low light intensity the PE-containing clones showed higher photosynthetic performance, larger photosynthetic units sizes, reaction center I to II ratios near unity, and steeper initial slopes of photosynthesis versus irradiance curves than a non-PE-containing clone. These findings demonstrate the high photosynthetic efficiency of PE-containing clones in low light environments common to middepth neritic and oceanic habitats.

Chloroplast nucleoids in a unicellular hot spring algaCyanidium caldarium and related algae

Algal chloroplast nucleoids were compared by epifluorescent microscopy.Cyanidium caldarium strain RK-1 or 001 has a rod-shaped chloroplast nucleoid whileCyanidium caldarium (Chroococcidiopsis sp.) strain M-8 or 002 has a circular chloroplast nucleoid along the periphery of a multilobed chloroplast.

Evolution of O2 in Brown Algal Chloroplasts

A METHOD IS DESCRIBED FOR THE ISOLATION OF PHOTOSYNTHETICALLY ACTIVE CHLOROPLASTS FROM FOUR SPECIES OF BROWN ALGAE: Fucus vesiculosis, Nereocystis luetkeana, Laminaria saccharina, and Macrocystis integrifolia. When compared to lettuce and spinach chloroplasts, the algal chloroplasts all showed lower activities for both photosystems II and I. Chloroplasts from all the plants produced H(2)O(2), with photosystem I functioning as the O(2) reductant in the light. In contrast to the green plants, however, brown algal chloroplasts strongly reduced O(2) under conditions where both photosystems II and I remain active. Relative variable fluorescence values were lower both in intact plants and chloroplasts of the brown algae than for either spinach or lettuce. It is suggested that although light harvesting activities appear similar in all the plants, details of electron transport in brown algae may differ from those of green plants.

THE DECONVULATION OF ABSORPTION SPECTRA OF GREEN PLANT MATERIALS—IMPROVED CORRECTIONS FOR THE SIEVE EFFECT

Abstract—The extinction (absorbance, optical density) of a suspension of algal cells or chloroplasts, etc. can be an a linear function of the absorption coefficient of the cell contents, even when the effects of scattering are negligible. The deviation from linearity is Duysens' sieve effect. The heights of absorption maxima of suspensions are depressed as compared with those of the “particle” contents. Duysens proposed an expression for correcting for this distortion; that expression proves to be a special approximate case of a more general one. This paper proposes an improved method of correction which utilizes this general expression, conventional expressions for particle absorption based on the Lorentz‐Mie and Aden and Kerker equations for scattering and absorbing, and iterative methods for numerically deducing particle absorption coefficients from suspension parameters. The procedure is illustrated by applying it to previous experimental spectra of Chlorellu cells and spinach chloroplasts.

DETERMINATION OF THE IN VIVO ABSORPTION AND PHOTOSYNTHETIC PROPERTIES OF THE LICHEN Acarospora schleicheri USING PHOTO ACOUSTIC SPECTROSCOPY

Abstract—Photoacoustic spectroscopy was used to determine the in vivo spectra of the stratified components of the lichen Acarospora schleicheri. There were three pigmented layers observed by the photoacoustic method: an absorption band associated with the pigment rhizocarpic acid, a bulk cytochrome absorption, and the absorption of the algal chloroplast. Due to the different absorption properties of these distinct layers, the photoacoustic technique was able to monitor the physical effect of hydration on the lichen. Hydration of the lichen reduced the scatter of the sample, increasing the effective incident light intensity within the sample. Hydration also resulted in an increase in the optical absorption coefficient of the algal layer, which was interpreted as a movement of the algal chloroplast in response to low light fluence and hydrated conditions.The photoacoustic technique was also used to monitor the relative photosynthetic activity of the algae within the lichen in a hydrated and dehydrated state. The photosynthetic assay could detect photosynthetic activity in the hydrated state but not in the dehydrated state. In addition, the photosynthetic response was found to be induced and repressed with light and dark periods respectively and its detection was limited at high frequencies by the damping of the thermal wave from the algal layer.

Cytoplasmic and chloroplast synthesis of phycobilisome polypeptides

In vivo labeling of eukaryotic phycobilisomes in the presence of inhibitors of translation on 70S and 80S ribosomes demonstrates that some of the polypeptides of this light-harvesting complex are synthesized in the cytoplasm while others are synthesized in the chloroplast. The major pigmented polypeptides, the alpha and beta subunits of the biliproteins (molecular weights between 15,000 and 20,000) and the anchor protein (molecular weight about 90,000) are translated on 70S ribosomes. This suggests that these polypeptides are made within the algal chloroplast. Because the alpha and beta subunits comprise a group of closely related polypeptides, the genes encoding these polypeptides may reside in the plastid genome as a multigene family. Other prominent phycobilisome polypeptides, including a nonpigmented polypeptide that may be involved in maintaining the structural integrity of the complex, are synthesized on cytoplasmic ribosomes. Because the synthesis of phycobilisomes appears to require the expression of genes in two subcellular compartments, this system may be an excellent model for: (i) examining interaction between nuclear and plastid genomes: (ii) elucidating the molecular processes involved in the evolution of plastid genes: (iii) clarifying the events in the synthesis and assembly of macromolecular complexes in the chloroplast.

Photosynthetic Unit Organization in a Red Alga

The relative concentration of biliproteins, phycobilisomes, chlorophyll a, and reaction centers I and II are reported for Neoagardhiella bailyei, a macrophytic red alga collected in the field and compared with Anacystis nidulans, a cyanobacterium cultured in the laboratory. The ratios of chlorophyll to reaction center I, of chlorophyll to reaction center II, and the mass of phycobiliprotein per reaction center II are quite similar in Neoagardhiella and Anacystis, supporting the concept that the red algal chloroplast is derived from a cyanobacterial progenitor. The ratios of reaction center I to reaction center II are about 2.3 in both species. The Anacystis phycobilisome has about 40% of the mass of the Neoagardhiella phycobilisome, 4.9 and 14.9 x 10(6) daltons, respectively. The reaction center II/phycobilisome ratio is about 1.7 in Anacystis and 4.1 in Neoagardhiella. Phycobilisome size and physical restrictions on phycobilisome packing may be a major constraint on the reaction center II-phycobilisome association and the assembly of the photosynthetic membrane in both the red algae and cyanobacteria.