Immunocytochemical Localization of Ribulose-1,5-Bisphosphate Carboxylase in the Pyrenoid and Thylakoid Region of the Chloroplast of Chlamydomonas reinhardtii

The distribution of the large and small subunits of ribulose-1,5-bisphosphate carboxylase in the chloroplast of Chlamydomonas reinhardtii was studied by immunoelectron microscopy by labeling Lowicryl-embedded sections with antibody to each subunit followed by protein A-gold. In light-harvested synchronously dividing cells, antibodies to each subunit heavily labeled the pyrenoid, whereas the thylakoid region of the plastid was lightly labeled. By estimating the volume of each chloroplast compartment, it was determined that approximately 40% of the total small subunit in the plastid and 30% of the large subunit are localized in the thylakoid region, presumably in the stroma. In synchronously dividing cells exposed to an extended dark period, the amount of labeling of the pyrenoid region by antibody to the small subunit stayed constant, but the labeling of the thylakoid region decreased. In stationary phase cells, the proportion of the label over the pyrenoid is higher than in synchronously dividing cells suggesting that the pyrenoid may be a storage organelle.     

Protein translocation into and within cyanelles (Review)

The cyanelles of the glaucocystophyte alga Cyanophora paradoxa resemble endosymbiotic cyanobacteria in morphology, pigmentation and, especially, in the presence of a peptidoglycan wall situated between the inner and outer envelope membranes. However, it is now clear that cyanelles in fact are primitive plastids. Phylogenetic analyses of plastid, nuclear and mitochondrial genes support a single primary endosymbiotic event. In this scenario cyanelles and all other plastid types are derived from an ancestral photosynthetic organelle combining the high plastid gene content of the Porphyra purpurea rhodoplast and the peptidoglycan wall of glaucocystophyte cyanelles. This means that the import apparatus of all primary plastids should be homologous. Indeed, heterologous in vitro import can now be shown in both directions, provided a phenylalanine residue essential for cyanelle import is engineered into the N-terminal part of chloroplast transit peptides. The cyanelle and likely also the rhodoplast import apparatus can be envisaged as prototypes with a single receptor showing this requirement for N-terminal phenylalanine. In chloroplasts, multiple receptors with overlapping and less stringent specificities have evolved explaining the efficient heterologous import of native precursors from C. paradoxa. With respect to conservative sorting in cyanelles, both the Sec and Tat pathways could be demonstrated. Another cyanobacterial feature, the dual location of the Sec translocase in thylakoid and inner envelope membranes, is also unique to cyanelles. For the first time, protease protection of internalized lumenal proteins could be shown for cyanobacteria-like, phycobilisome-bearing thylakoid membranes after import into isolated cyanelles.     

Properties of the Photosynthetic System and DNA of Cyanophora paradoxa Cyanelles

The cyanelle from the photosynthetic biflagellate protist Cyanophora paradoxa has been studied in terms of its photosynthetic properties. Structurally, the cyanelle resembles unicellular cyanobacteria. The cyanelle is readily released from the host cell by means of the French press. The isolated cyanelle shows typical photosystem I and photosystem II activities as well as phenazine methosulfate-mediated photophosphorylation. The kinetic parameters K_m and V_max were determined for CO₂ fixation in the cyanelle and cells of C. paradoxa and compared to a cyanobacterium. The determined values were not much different, although the cyanobacterium had a significantly greater rate of CO₂ fixation, and the cyanelle was least active in this regard. Photosystem I chlorophyll-protein complex is readily isolated from the thylakoid membrane. In all these respects, the photosynthetic apparatus of the cyanelle resembles that of cyanobacteria. No nitrogen fixation activity was observed. Attempts to regenerate the isolated cyanelle were not successful, but in some cases, an unidentified cyanobacterium grew up in standing cultures of C. paradoxa cyanelles. Buoyant density data indicate that the strain of C. paradoxa we have investigated differs from that employed by others, since our strain shows a value of 1.716 grams per cubic centimeter and others report values of 1.695 and 1.691.     

On the developmental dependence between Cyanophora paradoxa and Cyanocyta korschikoffiana in symbiosis

The prokaryote Cyanocyta korschikoffiana was isolated from the eukaryote Cyanophora paradoxa. The synthesis of several thylakoid proteins in these cyanelles is influenced by light and darkness and is sensitive to cycloheximide, the inhibitor of the eukaryotic host's translation. The possibility of a direct coordination between the translations of the host and of the cyanelles is discussed.Abbreviations CHM treatment addition of cycloheximide - CPN chlorophylline - PBN phycobiline - SDS-PAGE sodium-dodecylsulphate-polyacrylamide gelelectrophoresis     

Immunocytochemical localization of nitrite reductase in green algae

The distribution of nitrite reductase (EC 1.7.7.1) in the green algae Chlamydomonas reinhardtii, Monoraphidium braunii, Chlorella fusca, and Scenedesmus obliquus was studied by immunoelectron microscopy. The labeling of ultrathin cryosections was performed with anti-nitrite reductase antibodies followed by gold-labeled goat anti-rabbit antibodies. In C. reinhardtii sections, gold label was mainly associated with the pyrenoid, tonoplast, and plasmalemma. Significant labeling was also detected in the thylakoid region. In all other organisms, label density was lower but distributed in the same locations, except that the plasmalemma of S. obliquus was not significantly labeled. From estimates of the relative volume of different cell regions, we found that approximately 80% of the total enzyme is located in the chloroplastic region (thylakoids plus pyrenoid) of C. reinhardtii, M. braunii, and C. fusca, and 97% in the case of S. obliquus.     

Structure of the Thylakoids and Envelope Membranes of the Cyanelles of Cyanophora paradoxa

The cyanelles of Cyanophora paradoxa Korsch. are photosynthetically active obligate endosymbionts in which phycobiliproteins serve as the major accessory pigments. Freeze-fracture electron micrographs of thylakoids in isolated cyanelles reveal long parallel rows of particles covering most of the E-face, while a more random particle arrangement is evident in some areas. The center-to-center spacing of particles within these rows is about 10 nanometers. Their mean diameter was measured at 9.4 nanometers. The particles on the P-face have a mean diameter of 7.2 nanometers. Thylakoids that retained nearly the full complement of phycobiliproteins (determined spectrophotometrically and by gel electrophoresis) were isolated from the cyanelles. In thin sections of these preparations, rows of disc-shaped phycobilisomes are evident on the surface of the thylakoids. The spacing of the rows of phycobilisomes corresponds to that of the rows of E-face particles (approximately 45 nanometers, center to center). The periodicity of the disc-shaped phycobilisomes within a row is 10 nanometers suggesting a one-to-one association between phycobilisomes and E-face particles.

In addition, visualization of the protoplasmic surface (PS) of isolated thylakoids by freeze-etch electron microscopy shows that rows of disc-shaped phycobilisomes are aligned directly above rows of particles exhibiting two subunits, presumably the P-surface projections of the 10-nanometer intramembrane particles. These observations, together with earlier studies indicating that the 10-nanometer E-face particles probably represent photosystem II (PSII) complexes, suggest that phycobilisomes are positioned on the thylakoid surface in direct contact with PSII centers within the thylakoid membrane.

The inner envelope membrane of the cyanelles, observed in freeze-fracture replicas, resembles cyanobacterial plasma membranes and is dissimilar to the chloroplast envelope membranes of red or green algae. The envelope of isolated cyanelles exhibits two additional layers: (a) a 5- to 7-nanometer-thick layer that lies adjacent to the inner membrane and which seems to correspond to the peptidoglycan layer of cyanobacteria; and (b) a layer external to the purported peptidoglycan layer that exhibits fracture faces similar to those of the lipopolysaccharide layer of gram negative bacteria. Our findings indicate that the supramolecular architecture of cyanelles differs only slightly from free-living cyanobacteria to which they are presumably related.

     

小球藻Rubisco在叶绿体中的免疫金标定位

应用免疫技术对Rubisco在中国小球藻(Chlorellaspp.640909)叶绿体中进行了分子定位及Native-PAGE电泳、SDS-PAGE电泳及其Westen印迹分析,并对小球藻淀粉核(Pyrenoid)超微结构进行了观察.结果显示Native-PAGE电泳图谱主要为一条主带,Westen印迹反应证明该条带即为Rubisco酶,SDS-PAGE电泳及其Western印迹图谱显示Rubisco大亚基分子量大约为55kD.中国小球藻淀粉核为椭圆形,被淀粉鞘所包围,中央有一条由2个类囊体组成的纵向通道,并在蛋白核内段处稍膨胀.淀粉核与叶绿体基质存在多处联系.免疫分子定位显示Rubisco大亚基和全酶分子主要分布于叶绿体的淀粉核上,且Rubisco在淀粉鞘部位也有少量分布,极少部分分布在叶绿体基质中,表明叶绿体淀粉核与光合作用关系密切.Rubisco聚集于淀粉核可能有利于藻类对CO2固定.     

Immunogold localization of ribulose-1,5-bisphosphate carborylsae/oxygenase in chloroplasts of Chlorella]

Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a first key enzyme in the Calvin Circle of plant cell photosynthesis. This paper mainly studied gold immunolocalization of Rubisco of Chlorella spp. 640909, and the Native-PAGE and, SDS-PAGE and Western bloting analysis, as well as the observation to pyrenoid ultra structure. The Native-PAGE result showed a main band, evidenced as the Rubisco band by the Western blot with the antibody against the Rubisco from C. prototothecoides, The special immunoacton of Rubisco from Chlorella spp. 640909 and the antibody to large subunit of Rubisco from C. prothecoides showed the large subunit proteins of Rubisco in the two species of Chlorella shared the high homology. The SDS-PAGE and Western blotting maps showed the molecule weight of the large subunit of Rubisco of Chlorella spp. 640909 was about 55 KD. The shape of pyrenoid ultra structure of the electronic microscope was oblong, and was embedded in starch sheath, with 2 swelling thylakoids through out a center portrait channel of the pyrenoid. There were some connections between pyrenoid and the chloroplast stroma. The distribution of the large subunits and the whole Rubisco in the chloroplast of Chrolella spp. 640909 was studied by immunoelectron microscopy by embedded sections with antibody to large subunit and whole enzyme followed by second antibody, goad anti-rabbit immunoglobulin G conjugated to 10 nm gold particles(Sigma production). The result showed the antibodies against large subunit and whole enzyme heavily labeled the pyrenoid, as well as starch sheath region, whereas the thylakoid region of the plastid was lightly labeled. And the whole Rubisco antibody labeled the pyrenoid surface more heavily than the large subunit antibody did. It is demonstrated the pyrenoid and starch sheath have the photosynthesis function. Rubisco concentrating in pyrenoid and starch sheath is valuable to fix CO2 for photosynthesis in algae.     

Isolation of the plastid FtsZ gene from Cyanophora paradoxa (Glaucocystophyceae, Glaucocystophyta)

Plastids of glaucocystophytes are termed cyanelles and retain primitive features, such as a peptidoglycan wall. We isolated a full‐length prokaryotic plastid division gene, FtsZ, from the glaucocystophyte alga Cyanophora paradoxa Korshikov (CpFtsZ‐cy). CpftsZ‐cy has a chloroplast‐targeting signal at the N‐teminus. Immunofluorescence microscopy showed that CpFtsZ‐cy forms a ring‐like structure at the division plane of cyanelles.     

Transfer RNA gene mapping studies on cyanelle DNA from Cyanophora paradoxa

Summary The 4S RNA of cyanelles from Cyanophora paradoxa strain LB 555 UTEX was fractionated by two-dimensional gel electrophoresis. Individual tRNA species were identified by aminoacylation, labeled in vitro and hybridized to restriction endonuclease fragments of cyanelle DNA. Hybridization experiments, using individual tRNA species, have revealed the location of two tRNA genes, coding for tRNA^Ala and tRNA^Ile, in each of the two spacer segments separating the 16S and 23S rRNA genes on the two inverted repeats (10 kbp each) and three tRNA genes in the small single-copy region (17 kbp) separating the two inverted repeats. A minimum of 14 tRNA genes in the large single-copy region (88.5 kbp) has also been found.Heterologous hybridization studies, using cyanelle tRNAs and chloroplast DNA from spinach, broad bean, or maize, indicate a high degree of homology between some tRNAs from cyanelles and chloroplasts.Although cyanelles are often condisered as having evolved from endosymbiotic cyanobacteria, the organization of tRNA genes on cyanelle DNA and the results of heterologous hybridization studies show that cyanelles are related to higher plant chloroplasts.     

The Cyanelles of Cyanophora Paradoxa

The cyanelles of Cyanophora paradoxa, plastids surrounded by a peptidoglycan wall, are considered as a surviving example for an early stage of plastid evolution from endosymbiotic cyanobacteria. We highlight the model character of the system by focusing on three aspects: “organelle wall” structure, plastid genome organization, and protein translocation.

The biosynthetic pathway for cyanelle peptidoglycan appears to be analogous to that in Escherichia coli. Also, the basic structure of this peculiar organelle wall corresponds to that of the E. coli sacculus, with one notable exception: the C-1 carboxyl group of the D-isoglutamyl residue is partially amidated with N-acetylputrescine. Cyanelles harbor on their completely sequenced 135.6-kb genome genes for approximately 150 polypeptides, many of which are nucleus encoded in higher plants. Nevertheless, there are striking parallels in genome organization between cyanelles (and other primitive plastids) and higher plant chloroplasts. The transit sequences of nucleus-encoded cyanelle preproteins resemble stroma targeting peptides of higher plant chloroplast precursors. Heterologous import of precursors from C. paradoxa into isolated pea chloroplasts is possible and vice versa. Cyanelles are considered to represent a very early, diverging branch of plastid evolution and are derived from the semiautonomous endosymbiont that had already abandoned about 90% of its genetic information but still retained its prokaryotic wall. Recent data on the molecular biology of cyanelles and rhodoplasts are consistent with the assumption of a primary endosymbiotic event that was not only monophyletic with respect to the cyanobacterial invader, but also singular.

Cyanophora paradoxa is the best-investigated member of the glaucocystophyceae, phototrophic protists containing cyanelles, that is, plastids stabilized by a peptidoglycan-containing envelope. The classification of this group, comprising only eight (mostly monotypic) genera, is also based on parallels in morphology and organization of the “host cells” (Kies, 1992). Recently, this was corroborated by 16S and 18S rRNA-based phylogenetic analysis (Helmchen et al., 1995; Bhattacharya et al, 1995). Apart from C. paradoxa, only Glaucocystis nostochinearum can be grown at a reasonable rate. Thus, biochemical and molecular genetic data are mostly available for C. paradoxa and more precisely for the isolate 555UTEX (Pringsheim) that is kept in the major culture collections of algae. Biochemical work done on C. paradoxa and the sequencing of individual cyanelle genes have been described in several recent reviews (Schenk, 1992; Löffelhardt and Bohnert, 1994a,b). Here we discuss three topics: the cyanelle wall, aspects deduced from the complete cyanelle genome sequence, and protein translocation into and within cyanelles.     

Inorganic carbon acquisition by eukaryotic algae: four current questions

The phylogenetically and morphologically diverse eukaryotic algae are typically oxygenic photolithotrophs. They have a diversity of incompletely understood mechanisms of inorganic carbon acquisition: this article reviews four areas where investigations continue. The first topic is diffusive CO₂ entry. Most eukaryotic algae, like all cyanobacteria, have inorganic carbon concentrating mechanisms (CCMs). The ancestral condition was presumably the absence of a CCM, i.e. diffusive CO₂ entry, as found in a small minority of eukaryotic algae today; however, it is likely that, as is found in several cases, this condition is due to a loss of a CCM. There are a number of algae which are in various respects intermediate between diffusive CO₂ entry and occurrence of a CCM: further study is needed on this aspect. A second topic is the nature of cyanelles and their role in inorganic carbon assimilation. The cyanelles (plastids) of the euglyphid amoeba Paulinella have been acquired relatively recently by endosymbiosis with genetic integration of an α-cyanobacterium with a Form 1A Rubisco. The α-carboxysomes in the cyanelles are presumably involved in a CCM, but further investigation is needed.Also called cyanelles are the plastids of glaucocystophycean algae, but is it now clear that these were derived from the β-cyanobacterial ancestor of all plastids other than that of Paulinella. The resemblances of the central body of the cyanelles of glaucocystophycean algae to carboxysomes may not reflect derivation from cyanobacterial β-carboxysomes; although it is clear that these algae have CCMs but these are now well characterized. The other two topics concern CCMs in other eukaryotic algae; these CCMs arose polyphyletically and independently of the cyanobacterial CCMs. It is generally believed that eukaryotic algal, like cyanobacterial, CCMs are based on active transport of an inorganic carbon species and/or protons, and they have C₃ biochemistry. This is the case for the organism considered as the third topic, i.e. Chlamydomonas reinhardtii, the eukaryotic alga with the best understood CCM. This CCM involves HCO₃ ^? conversion to CO₂ in the thylakoid lumen so the external inorganic carbon must cross four membranes in series with a final CO₂ effux from the thylakoid. More remains to be investigated about this CCM. The final topic is that of the occurrence of C₄-like metabolism in the CCMs of marine diatoms. Different conclusions have been reached depending on the organism investigated and the techniques used, and several aspects require further study.     

The dynamic surface of dividing cyanelles and ultrastructure of the region directly below the surface in <Emphasis Type="Italic">Cyanophora paradoxa</Emphasis>

The cyanelles of glaucocystophytes are probably the most primitive of known extant plastids and the closest to cyanobacteria. Their kidney shape and FtsZ arc during the early stage of division define cyanelle division. In order to deepen and expand earlier results (Planta 227:177–187, 2007), cells of Cyanophora paradoxa were fixed with two different chemical and two different freeze-fixation methods. In addition, cyanelles from C. paradoxa were isolated to observe the surface structure of dividing cyanelles using field emission scanning electron microscopy (FE-SEM). A shallow furrow started on one side of the division plane. The furrow subsequently extended, covering the entire division circle, and then invaginated deeply, becoming clearly visible. The typical FtsZ arc was 2.3–3.4 μm long. This length matches that of the cleavage furrow observed using FE-SEM. The cyanelle cleavage furrows are from one-fourth to one-half of the circumference of the division plane. The shallow furrow that appears on the cyanelle outer surface effectively changes the division plane. Using freeze-fixation methods, the electron-dense stroma and peptidoglycan could be distinguished. In addition, an electron-dense belt structure (the cyanelle ring) was observed inside the leading edge at the cyanelle division plane. The FtsZ arc is located at the division plane ahead of the cyanelle ring. Immunogold-TEM localization shows that FtsZ is located interiorly of the cyanelle ring. The lack of an outer PD ring, together with the arch-shaped furrow, suggests that the mechanical force of the initial (arch shaped) septum furrow constriction comes from inside the cyanelle.     

Effect of dissolved inorganic carbon on the expression of carboxysomes,localization of Rubisco and the mode of inorganic carbon transport in cells of the cyanobacterium Synechococcus UTEX 625

In the cyanobacterium Synechococcus UTEX 625, the extent of expression of carboxysomes appeared dependent on the level of inorganic carbon (CO₂+HCO _inf3 ^sup- ) in the growth medium. In cells grown under 5% CO₂ and in those bubbled with air, carboxysomes were present in low numbers (<2 · longitudinal section^-1) and were distributed in an apparently random manner throughout the centroplasm. In contrast, cells grown in standing culture and those bubbled with 30 l CO₂ · 1^-1 possessed many carboxysomes (>8 · longitudinal section^-1). Moreover, carboxysomes in these cells were usually positioned near the cell periphery, aligned along the interface between the centroplasm and the photosynthetic thylakoids. This arrangement of carboxysomes coincided with the full induction of the HCO _inf3 ^sup- transport system that is involved in concentrating inorganic carbon within the cells for subsequent use in photosynthesis. Immunolocalization studies indicate that the Calvin cycle enzyme ribulose bisphosphate carboxylase was predominantly carboxysome-localized, regardless of the inorganic carbon concentration of the growth medium, while phosphoribulokinase was confined to the thylakoid region. It is postulated that the peripheral arrangement of carboxysomes may provide for more efficient photosynthetic utilization of the internal inorganic carbon pool in cells from cultures where carbon resources are limiting.Abbreviations Chl chlorophyll - DIC dissolved inorganic carbon (CO₂+HCO _inf3 ^sup- +CO _inf3 ^sup2- ) - PRK phosphoribulokinase - RuBP ribulose 1,5-bisphosphate - Rubisco LS large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase     

In Vivo and In Vitro Protein Phosphorylation Studies on Ochromonas danica, an Alga with a Chlorophyll a/c/Fucoxanthin Binding Protein

The phosphorylation of thylakoid membranes in the Chromophyte alga Ochromonas danica was studied in whole cells and in vitro. Protein kinase activity was observed in the thylakoid fraction, and several membrane-bound polypeptides were found to be phosphorylated. The thylakoid protein kinase demonstrated several unusual regulatory properties. Both the polypeptides that were phosphorylated and the rate of protein phosphorylation were independent of illumination. Protein kinase activity was also unaffected by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, diuron. The kinase activity was inhibited under strong reducing conditions. Whole cells labeled with ³²PO₄³⁻ were converted to light states I and II by pre-illumination favoring photosystem I or photosystem II, respectively. Analysis of the phosphoproteins from cells in state I and state II showed that no changes in phosphorylation accompanied the change in energy redistribution.     

Exchange of Metabolites in Cyanophora paradoxa and its Cyanelles*

The flagellate Cyanophora paradoxa contains blue-greenish, organelle-like inclusions termed cyanelles which perform photosynthetic CO₂-fixation in place of chloroplasts. By the use of the HPLC-technique, Cyanophora was shown to form glucose, sucrose, maltose, mannitol, ribose, glycerol and trehalose. Extracts from the whole organism and from the eucaryotic host, but not from the cyanelles, convert ¹⁴C-labelled UDP-glucose to polyglucan. Synthesis of sucrose from UDP-glucose and fructose-6-P or fructose could not be demonstrated in any extract from Cyanophora. The transfer of metabolites into cyanelles was monitored by the silicone oil filtering technique. The solute spaces for ¹⁴C-labelled sorbitol and ³H₂O were the same indicating that sorbitol freely penetrated the plasma membrane of cyanelles in contrast to the situation found in chloroplasts. The measurements of the solute spaces for the different compounds showed that maltose and sucrose were not accumulated by isolated cyanelles. Other compounds like fructose, fucose, glutamine or glycine had intermediate sizes of their solute spaces. Cyanelles apparently possess a rapidly transporting glucose carrier and not a malate/oxaloacetate shuttle and also not an ATP/ADP translocator. The carrier composition at the plasma membrane of cyanelles and at the inner envelope membrane of chloroplasts seems to be totally different.     

CYTOCHEMICAL STUDIES ON PROCHLOROPHYTES: LOCALIZATION OF DNA AND RIBULOSE 1,5-BISPHOSPHATE CARBOXYLASE-OXYGENASE1

We studied the distribution of the DNA-containing region and the ribulose 1,5-bisphosphate carboxylase-oxygenase (RuBisCo) content of polyhedral bodies in three different prochlorophyte cell types in a search for broad evolutionary affinities of these chlorophyll b-containing prokaryotes. DNA was localized by DAPI staining and electron microscopy utilizing monoclonal anti-DNA antibody 2C-10 plus a secondary antibody labeled with colloidal gold. Antibodies against the large RuBisCo subunit from a higher plant raised in rabbits were used to localize RuBisCo in polyhedral bodies. We studied Prochloron Lewin cells from two different didemnid ascidian hosts (Lissoclinum patella and Didemnum molle) collected in Palau, West Caroline Islands, and cells of Prochlorothrix hollandica Burger-Wiersma, Stal, and Mur grown in laboratory culture. Cells of the blue-green alga Anabaena 7120 were studied for comparison. The DNA distribution was markedly different in the two Prochloron cell types. The thylakoids in cells from L. patella were concentrically arranged around a large central vacuole; the DNA-containing stromal areas appeared in thin sections as a concentric arcs between the thylakoid stacks. The central vacuole was lacking in cells from D. molle, and the thylakoid stacks and strands of DNA-containing stroma showed a more haphazard arrangement. In the filamentous Prochlorothrix the DNA-containing stroma was largely limited to a central nucleoid structure running the length of the cell. Although the DNA arrangements in Prochloron might be considered “chloroplast-like” since DNA-containing stroma is distributed, as in chloroplasts, in scattered sites among photosynthetic membranes, this is not so in Prochlorothrix, where there is an axial nucleoid, as in many filamentous cyanobacteria. Our anti-RuBisCo antibodies were selectively bound to the polyhedral bodies of all three cell types, indicating that Prochloron and Prochlorothrix, like many other autotrophic prokaryotes, possess typical carboxysomes.     

Metabolic activities in Cyanophora paradoxa and its cyanelles

Isolated cyanelles of Cyanophora paradoxa perform photosystem I and II dependent Hill reactions. The photosynthetic electron transport of the cyanelles does not show special features uncommon in cyanobacteria or chloroplasts of red algae. A preparation of cyanelles performs photosynthetic O₂-evolution with approximately 1/3 of the rate of intact Cyanophora, in only, however, the first three minutes of the experiment. All attempts to stabilize the CO₂-fixation activity of isolated cyanelles failed. Isolated cyanelles do not perform KCN-sensitive O₂-uptake, indicating that respiratory cytochrome oxidase is lacking in cyanelles. O₂-consumption by crude extracts from Cyanophora is inhibited by KCN when N-tetramethyl-p-phenylenediamine/ascorbate or NADH but not NADPH are supplied as the electron donors in contrast to the situation in cyanobacteria. These findings suggest that cyanelles do not respire. It is concluded that cyanelles are not so much related to cyanobacteria as formerly believed, but share many properties with chloroplasts of eukaryotic cells.Abbreviations Chl chlorophyll - DCPIP dichlorophenol-indophenol - TMPD N-tetramethyl-p-phenylenediamine To whom correspondence should be addressed     

Conserved relationship between FtsZ and peptidoglycan in the cyanelles of Cyanophora paradoxa similar to that in bacterial cell division

Cyanelles of the biflagellate protist Cyanophora paradoxa have retained the peptidoglycan layer, which is critical for division, as indicated by the inhibitory effects of β-lactam antibiotics. An FtsZ ring is formed at the division site during cyanelle division. We used immunofluorescence microscopy to observe the process of FtsZ ring formation, which is expected to lead cyanelle division, and demonstrated that an FtsZ arc and a split FtsZ ring emerge during the early and late stages of cyanelle division, respectively. We used an anti-FtsZ antibody to observe cyanelle FtsZ rings. We observed bright, ring-shaped fluorescence of FtsZ in cyanelles. Cyanelles were kidney-shaped shortly after division. Fluorescence indicated that FtsZ did not surround the division plane at an early stage of division, but rather formed an FtsZ arc localized at the constriction site. The constriction spread around the cyanelle, which gradually became dumbbell shaped. After the envelope’s invagination, the ring split parallel to the cyanelle division plane without disappearing. Treatment of C. paradoxa cells with ampicillin, a β-lactam antibiotic, resulted in spherical cyanelles with an FtsZ arc or ring on the division plane. Transmission electron microscopy of the ampicillin-treated cyanelle envelope membrane revealed that the surface was not smooth. Thus, the inhibition of peptidoglycan synthesis by ampicillin causes the inhibition of septum formation and a marked delay in constriction development. The formation of the FtsZ arc and FtsZ ring is the earliest sign of cyanelle division, followed by constriction and septum formation.     

Novel insights into the biopharmaceutical potential,comparative phytochemical analysis and multivariate analysis of different extracts of shea butter tree -Vitellaria paradoxa C. F. Gaertn

Vitellaria paradoxa C. F. Gaertn. (Family: Sapotaceae), is a well-known medicinal plant but with no consolidated published literature to substantiate its traditional uses. This research aimed to investigate the pharmaceutical potential of V. paradoxa extracts and attempts to compare the biological profiles and phytochemical analysis prepared by different extraction protocols. For preparation of the V. paradoxa leaves and stem bark extracts, four extraction techniques and two different solvent types were employed. First analysis included identification of bioactive compounds by use of high-resolution Quadrupole Time-Of-Flight instrument. Antioxidant capacities were evaluated as radical scavenging potential, reducing potential, total antioxidant capacity (phosphomolybdenum) and metal chelating power. The last evaluation step included analysis of the inhibitory properties of V. paradoxa extracts against key enzymes related to main health problems. Our findings revealed key details on phytochemical profiling of the V. paradoxa plant, whereas 17 phytochemicals were identified in leaves and 14 in stem bark. Antioxidant assays showed that extracts obtained by maceration extraction process exhibit potent antioxidant capacities. Extracts prepared by HAE generally showed the highest enzymatic activities. The presented findings confirmed the need for further studies geared towards discovery and development of novel bioactive components from V. paradoxa leaves and stem bark.