THE PYRENOID OF SCENEDESMUS QUADRICAUDA

The development of the pyrenoid of Scenedesmus quadricauda from the time of its initiation and its subsequent activities is described in some detail. Correlation is made between the evidence from light and electron microscopy. The pyrenoid is a dynamic organelle which continues to change its appearance throughout the development of the algal cell due to the following factors: the deposition of starch platelets within the periphery of the expanding matrix; the separation of starch grains into individual pockets by the intrusive growth of the chloroplast lamellae in centripetal fashion; and the transition of the shape of the starch from concavo-convex platelets to lenticular grains. By these processes starch grains are continuously formed by deposition of carbohydrates within the matrix. The grains accumulate within the chloroplast, maintaining an organic connection with each other by slender starch bridges. Some parental starch grains are passed on to daughter cells during cell division. By taking into account the planes of cleavage during cell division, it is not difficult to see that pyrenoid starch grains could become distributed throughout the daughter chloroplast, regardless of their distance from the pyrenoid.     

STRUCTURE AND FUNCTION OF THE ALGAL PYRENOID. I. ULTRASTRUCTURE AND CYTOCHEMISTRY DURING ZOOSPOROGENESIS OF TETRACYSTIS EXCENTRICA1

The fine structure of the pyrenoid in the mature vegetative cell of Tetracystis excentrica Brown and Bold is described. During zoosporogenesis, the pyrenoid undergoes regression, and the ultrastructure of this process is described in detail. The ground substance undergoes dissolution, and reticulate fibrillar structures appear as well as intruding chloroplast thylakoids. Pyrenoid-associated starch plates diminish, and quantities of starch not associated with the pyrenoid are produced. New pyrenoids appear late in the division cycle after all other major organelles associated with the motile cell have been formed. Zoospore pyrenoids develop in thylakoid-free spaces of the chloroplast which are similar to the DNA-containing regions. The new pyrenoid ground substance, which is loosely fibrillar, arises in close proximity to starch grains which may be formed in the stroma. Then the zoospore pyrenoid produces 2 hemispherical starch plates identical to those in the mature vegetative cell. Zoospore pyrenoids lack the 2 convoluted thylakoids between the starch plates and the ground substance characteristic of those in the mature vegetative cell. Instead, the thylakoids are identical to those of the chloroplast at first, and then develop into a convoluted state in the vegetative cell. Cytochemical tests for DNA, RNA, and protein were made for the cytoplasm, nucleus, nucleolus, and pyrenoid. Conclusive evidence is presented for the presence of RNA in the cytoplasm and nucleolus, DNA in the nucleus, and protein in the pyrenoid. The tests did not conclusively demonstrate the presence or absence of DNA and RNA in the pyrenoid; however, they suggested that small amounts of both DNA and RNA may be present.     

The compound internal pyrenoid in cultured cells of the green algaMonoraphidium griffithii (Berkel.) Komar.-Legner.

Summary The chloroplast ultrastructure ofMonoraphidium griffithii (Berkel.) Komar.-Legner. has been studied in axenic cultures of various ages. The algae have grown in a complete nutrient solution (illumination about 3,000 lx) and on its agar medium (illumination about 600 lx).The large parietal cup-shaped chloroplast of the cells includes a multiformed compound internal pyrenoid that is situated, especially in older cells, in the central part of the chloroplast opposite to the dictyosome and the nucleus. The chloroplast thylakoids either reach the edge of the pyrenoid or penetrate its matrix and run there parallel in more or less long bits. Starch grains were not found to form any sheath around the pyrenoid regions. The number of starch grains increased with the age of the cell.     

几种藻类蛋白核的超微结构研究

应用电镜及免疫电镜技术对莱茵藻、小球藻、条浒苔和紫菜等藻类的叶绿体蛋白核的超微结构及主要组成成分进行了观察和研究。结果显示：不同藻类的蛋白核结构不同,显示了藻类蛋白核的多样性。蛋白核为球形或椭圆形,由蛋白质组成。莱茵藻、小球藻和条浒苔的蛋白核外围被淀粉鞘所包围,而紫菜的蛋白核外围无淀粉鞘而直接被叶绿体的类囊体所包围。淀粉鞘由淀粉组成,淀粉鞘的厚薄与藻体藻龄及增养状态有关系。在蛋白核中央,一般都具有由类囊体形成的孔道,使蛋白核与外界联系,小球藻和条浒苔蛋白核具有1条纵向孔道,而莱茵藻和紫菜为多条孔道。金相免疫技术检测结果显示Rubisco和Rubisco活化酶均在蛋白核及淀粉鞘区域中定位,表明蛋白核具有光合作用功能．     

Electron Microscopic Observations on the Symbiosis of Paramecium bursaria and its Intracellular Algae*

SYNOPSIS. Observations were made on the fine structure of Paramecium bursaria and its intracellular Chlorella symbionts. Emphasis was placed on the structure of the algae and structural aspects of the relationship between the organisms. The algae are surrounded by a prominent cell wall and contain a cup-shaped chloroplast which lies just beneath the plasma membrane. Within the cavity formed by the chloroplast are a large nucleus, a mitochondrion, one or more dictyosomes, and numerous ribosomes. The chloroplast itself is made up of a series of lamellar stacks each containing 2–6 or more thylakoids with a granular stroma and starch grains intercalated between the stacks. The thylakoid stacks of mature algae are frequently more compact than those of recently divided algae. A large pyrenoid is located within the base of the chloroplast. It is made up of a granular or fibrillar matrix surrounded by a shell of starch. The matrix is bisected by a stack of 2 thylakoids. Prior to the division of the chloroplast the pyrenoid regresses; pyrenoids subsequently form in the daughter chloroplasts thru condensation of the matrix material and the reappearance of a starch shell. This shell appears to be formed by the hollowing-out of starch grains already present in the chloroplast stroma. Accordingly, in this case, starch moves from the stroma to the pyrenoid. The algae are located thruout the peripheral cytoplasm of the Paramecium. Each alga is located in an individual vacuole except immediately following division of the algae when the daughter cells are temporarily located in the vacuole which harbored the parental cell. Shortly thereafter the vacuole membrane invaginates, thereby isolating the daughter algae into individual vacuoles. Degenerating symbiotic algae are seen; because these are frequently found in vacuoles with bacteria, they are presumed to be undergoing digestion. Due to the conditions of culture these algae could have been either of intracellular or extracellular origin.     

LIGHT AND ELECTRON MICROSCOPY OF PYRENOIDS AND SPECIES DELIMITATION IN VOLVULINA (CHLOROPHYTA,VOLVOCACEAE)1

The appearances of pyrenoids in the vegetative cells of Volvulina steinii Playfair and V. pringsheimii Starr were observed in detail by light and electron microscopy in relation to the culture age to clarify the taxonomic relationship between the two species. In V. pringsheimii, the pyrenoids were always present in the bottom of the cupshaped chloroplasts and their gross morphology did not vary in relation to the culture age, while those of V. steinii appeared de novo and developed as the culture aged. In 24-h cultures of V. steinii, pyrenoids were not observed in the chloroplasts. In 48-h cultures, a pyrenoid matrix developed apparently de novo in the brim of the cupshaped chloroplast. Subsequently, starch grains appeared around the pyrenoid matrix in 72-h cultures. The volume of the matrix and the associated starch grains increased and tubular channels entered into the pyrenoid matrix in 96-h cultures. In addition, the pyrenoid in the parental chloroplast of V. pringsheimii divided and was distributed to each daughter cell during cell divisions in daughter colony formation, while the parental pyrenoid of V. steinii did not divide and went to one of the daughter cells. Therefore, these two species can be clearly distinguished by the differences in the position of pyrenoids in the cupshaped chloroplasts and stability of pyrenoid appearance in relation to the culture age, as well as in the fate of parental pyrenoids during daughter colony formation.     

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.     

PHYLOGENETIC RELATIONSHIPS OF CAULERPA (CHLOROPHYTA) BASED ON COMPARATIVE CHLOROPLAST ULTRASTRUCTURE1,2

The ultrastructure of chloroplasts from 28 of the 73 species of Caulerpa Lamouroux (Chlorophyta, Caulerpales) has been studied to aid in interpreting phylogenetic relationships among the 12 recognized sections. Variations of systematic value include pyrenoid occurrence and fine structure, thylakoid architecture and amount of photosynthate storage. Comparisons of field and culture specimens indicate these characters are consistent. Chloroplast thylakoids are grouped into bands, with the distribution of bands differing among species. In the most common arrangement, bands are evenly distributed throughout the chloroplast. A few species show lateral displacement of bands whereas others have a majority of bands arranged at one end of the chloroplast. Starch is stored cither as one or two large grains (> 1 μm diam.) or numerous small grains (< 0.5 μm diam.). Electron-transparent regions are common in other species in which chloroplasts rarely store starch. Simple, embedded pyrenoids are present in several species of section Sedoideae. An opaque region occurs in chloroplasts of C. elongata which may represent an intermediate stage in the evolutionary loss of the pyrenoid. It is suggested that the chloroplast of Caulerpa evolved, from a large, complex, pyrenoid-containing organelle housing both photosynthetic and amylogenic functions, to a small, structurally simpler one, specialized for photosynthesis alone. A phylogeny of the 12 sections of Caulerpa is constructed, based on chloroplast evolution which agrees with an earlier morphology-based hypothesis on the origin and evolution of Caulerpa.     

ULTRASTRUCTURE OF THE CONCHOCELIS STAGE OF THE MARINE RED ALGA PORPHYRA LEUCOSTICTA1

The ultrastructure of the Conchocelis or filamentous stage of Porphyra leucosticta was investigated. Each cell contains 1 or 2 parietal, stellate chloroplasts with a single pyrenoid in each chloroplast. The centrally located nucleus is irregularly shaped and contains 1–2 nucleoli. The cytoplasm has typical floridean starch grains and nonmernbrane-bound lipid bodies. The cell wall is divided into an outer and an inner wall. Many lomasomes are associated with the cell membrane. Pit connections are found between cells, and their taxonomic significance is discussed.     

Über denChlorella-Phycobionten vonTrapelia coarctata

Trapelia coarctata is lichenized withChlorella saccharophila var.ellipsoidea; this is in accordance with one of two former statements. The cells of the isolated alga may be covered individually by a gelatinous envelope; they also can be embedded in confluent mucilage. The course of succedanous divisions leading to the formation of autospores starts with the appearance of a second, new pyrenoid and goes on with bipartioning of the chloroplast, nuclear division and cytokinesis. Starch grains identical to those in the stroma surround the pyrenoid more or less loosely and not in the form of saucer-shaped parts constituting a coherent shell.

     

Ultrastructure of Endosymbiotic Chlorella in a Vorticella

SYNOPSIS Observations were made on the ultrastructure of a species of Vorticella containing endosymbiotic Chlorella. The Vorticella , which were collected from nature, bore conspicuous tubercles of irregular size and distribution on the pellicle. Each endosymbiotic algal cell was located in a separate vacuole and possessed a cell wall and cup-shaped chloroplast with a large pyrenoid. The pyrenoid was bisected by thylakoids and surrounded by starch plates. No dividing or degenerating algal cells were observed.     

STRUCTURE AND DEVELOPMENT OF THE CHLOROPLAST IN CHLAMYDOMONAS : I. THE NORMAL GREEN CELL

The cytoplasmic organization of a normal green strain of the alga Chlamydomonas reinhardi has been investigated with the electron microscope using thin sections of OsO₄ fixed material. The detailed organization of the chloroplast has been of special interest. The chloroplast, a cup-shaped organelle, surrounded by a double membrane, consists of: (1) discs about 1 micron in diameter, considered to represent the basic structural unit of the chloroplast, and each composed of a pair of membranes joined at their ends to form a flat closed vesicle; the discs are grouped into stacks resembling the grana of higher plants; (2) matrix material of low density in which the discs are embedded; (3) starch grains; (4) the pyrenoid, a non-lamellar region associated with starch synthesis, and containing tubules which connect with the lamellae; (5) the eyespot, a differentiated region containing two or three plates of hexagonally packed, carotenoid-containing granules, located between discs, and associated with phototaxis. In addition to the chloroplast, the cytoplasm contains various membranous and granular components, including mitochondria, endoplasmic reticulum, and dictyosomes, identified on the basis of morphological comparability with structures seen in animal cells. The nucleus, not investigated in detail in this study, contains a large, granular nucleolus and is surrounded by a nuclear envelope which is provided with pores and exhibits instances of continuity with the endoplasmic reticulum of the cytoplasm.     

Structure and development of the chloroplast in Chlamydomonas. I. The normal green cell

The cytoplasmic organization of a normal green strain of the alga Chlamydomonas reinhardi has been investigated with the electron microscope using thin sections of OsO(4) fixed material. The detailed organization of the chloroplast has been of special interest. The chloroplast, a cup-shaped organelle, surrounded by a double membrane, consists of: (1) discs about 1 micron in diameter, considered to represent the basic structural unit of the chloroplast, and each composed of a pair of membranes joined at their ends to form a flat closed vesicle; the discs are grouped into stacks resembling the grana of higher plants; (2) matrix material of low density in which the discs are embedded; (3) starch grains; (4) the pyrenoid, a non-lamellar region associated with starch synthesis, and containing tubules which connect with the lamellae; (5) the eyespot, a differentiated region containing two or three plates of hexagonally packed, carotenoid-containing granules, located between discs, and associated with phototaxis. In addition to the chloroplast, the cytoplasm contains various membranous and granular components, including mitochondria, endoplasmic reticulum, and dictyosomes, identified on the basis of morphological comparability with structures seen in animal cells. The nucleus, not investigated in detail in this study, contains a large, granular nucleolus and is surrounded by a nuclear envelope which is provided with pores and exhibits instances of continuity with the endoplasmic reticulum of the cytoplasm.     

Effects of Light and Dark on the Ultrastructure of Lichen Algae

Quantitative ultrastructural observations have been made onthe algal cells (Trebouxia) in two lichens, Parmelia sulcataand P. laevigata, stored for 48 h in the dark or under a light/darkregime. The response of the alga was found to differ in theselichens. In P. sulcata the dark treatment caused a decreasein starch grains, lipid-rich pyrenoglobuli and peripheral cytoplasmicstorage bodies and an increase in pyrenoid and chloroplast proteinbodies. The algae in P. laevigata contained little starch andno chloroplast protein bodies. However, after dark treatment,starch, cytoplasmic storage bodies and pyrenoid dimensions sometimesdeclined, while pyrenoglobuli numbers increased. Some of theseapparent changes depended upon the units used for calculatingthe cross-sectional areas of structures, e.g. absolute units,percentage of cell wall, protoplast or chloroplast cross-sectionalarea. Chloroplast area increased in the dark (as a % of cellwall area) in both species while mitochondria were larger inthe dark in P. sulcata but not in P. laevigata. Ultrastructuralchanges were not clearly correlated with changes in photosyntheticand respiratory rates. These results directly support the suggestionthat some intra-cellular structures are energy-generating reservesthe dimensions of which can rapidly change. Parmelia sulcata, Parmelia laevigata, lichen algae, light and dark storage, starvation, reserve substances, organdie dimensions     

Ultrastructure of Characiochloris acuminata Lee et Bold

The ultrastructure of the vegetative cell and zoospore of Characiochloris acuminata Lee et Bold (Chlorangiellaceae, Tetrasporales, Chlorophyceae) is described.

The vegetative cell is distinctive in having numerous contractile vacuoles which are randomly distributed in the cytoplasm and visible through the fissures of the parietal chloroplast. A single pyrenoid, embedded in the chloroplast, is penetrated by cytoplasmic canals which are lined by the chloroplast envelope. The vegetative cell is attached to the substrate or host by two flagellar remnants (retained from the zoospore stage), each of which is ensheathed in a gelatinous tube through the cell wall at the cell base. The basal bodies are apparently abscissed from the flagellar shaft by a unit membrane which becomes continuous with the plasma membrane.

The zoospore is biflagellate, with the flagella equal in length, smooth and longer than the cell body. The flagellar sheath is characteristically undulate and the two flagellar bases are connected by a dense interflagellar fibre. The large nucleus has a conspicuously inflated nuclear envelope and the pyrenoid is similar to that of the vegetative cell.     

Changes of Starch Localization within the Chloroplast Induced by Changes in CO2 Concentration during Growth of Chlamydomonas reinhardtii: Independent Regulation of Pyrenoid Starch and Stroma Starch

To understand the physiological function of the pyrenoid, aprotein complex in algal chloroplast stroma with surroundingstarch sheaths, the effects of environmental conditions on thepyrenoid and pyrenoid starch were investigated in the unicellulargreen alga Chlamydomonas reinhardtii. Pyrenoid starch was rapidlyaccumulated within 5 hours when the extracellular CO₂ concentrationwas lowered from 4% to ordinary air level (0.04%). Startingwith high-CO₂ grown cells containing well-developed stroma starchgranules, degradation of stroma starch and accumulation of pyrenoidstarch were observed in parallel during the adaptation to lowCO₂ condition. This pyrenoid-starch accumulation was light dependentand completely inhibited by DCMU. The starch relocalizationprocess was reversible, but the breakdown of pyrenoid starchwas slower than its accumulation. The time courses of accumulation(or degradation) of starch around the pyrenoid paralleled increases(or decreases) in carbonic anhydrase (EC 4.2.1.1 [EC] ) activity,and the pyrenoid starch accumulation is thought to be one ofthe adaptation phenomena to CO₂ concentration. When nitrogenassimilation was inhibited, stroma starch and total starch contentincreased, while that of pyrenoid starch decreased. These resultsindicate that the synthesis and degradation of the two formsof starch were regulated independently by the environmentalconditions. ADP-glucose starch synthase (EC 2.4.1.21 [EC] ) activitywas detected at a physiological level, but the change of starchlocalization could not be explained by total starch synthaseactivity nor by starch-degrading enzyme activities. We assumethat starch metabolism around the pyrenoid is regulated independentlyfrom that in other stromal spaces. (Received May 16, 1988; Accepted July 17, 1988)     

The induction of the CO2-concentrating mechanism is correlated with the formation of the starch sheath around the pyrenoid of Chlamydomonas reinhardtii

The pyrenoid is a prominent proteinaceous structure found in the stroma of the chloroplast in unicellular eukaryotic algae, most multicellular algae, and some hornworts. The pyrenoid contains the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase and is sometimes surrounded by a carbohydrate sheath. We have observed in the unicellular green alga Chlamydomonas reinhardtii Dangeard that the pyrenoid starch sheath is formed rapidly in response to a decrease in the CO₂ concentration in the environment. This formation of the starch sheath occurs coincidentally with the induction of the CO₂-concentrating mechanism. Pyrenoid starch-sheath formation is partly inhibited by the presence of acetate in the growth medium under light and low-CO₂ conditions. These growth conditions also partly inhibit the induction of the CO₂-concentrating mechanism. When cells are grown with acetate in the dark, the CO₂-concentrating mechanism is not induced and the pyrenoid starch sheath is not formed even though there is a large accumulation of starch in the chloroplast stroma. These observations indicate that pyrenoid starch-sheath formation correlates with induction of the CO₂-concentrating mechanism under low-CO₂ conditions. We suggest that this ultrastructural reorganization under lowCO₂ conditions plays a role in the CO₂-concentrating mechanism C. reinhardtii as well as in other eukaryotic algae.     

Ultrastructural changes during normal and colchicine-inhibited cell division ofChlorella

Summary In synchronously growingChlorella strain 211-8 b protoplast division, i.e. cell plate formation ensued about 14 h after beginning of the light period, when cells under favourable growth conditions had already become tetranucleate. Initially, small electron transparent droplets appeared in a plane between the nuclei. They soon fused into a thin cell plate extending towards the cell periphery. The second and third plate began to be formed before the first was completed, and finally a continuous system of cell plates resulted, which obtained a considerable thickness by the 19th hour. Then, within a short time, all 8 or 16 protoplasts were simultaneously covered by a typical cell wall outer zone, and subsequently inner zones were formed of material derived from the cell plate and the inner zone of the wall of the mother cell. The remaining original outer zone broke at about the 22nd hour, thereby releasing the autospores.Addition of colchicine after 13 1/2 h of illumination caused a marked delay of the cell development and prevented cell division by almost completely disturbing the orientation of the developing cell plates. The polyploid and polynucleate protoplasts finally formed several outer zones below the cell wall which subsequently were translocated through the inner zone towards the cell surface where they partly became detached.The thick starch layers of the pyrenoid were degraded between the 10th and 17th hour, but the extra-pyrenoidal starch grains remained untouched. The undivided pyrenoid was transmitted during cell plate formation to one of the daughter cells where it finally degenerated. One small pyrenoid appeared in each daughter cell immediately after the cell walls had been formed. They soon obtained small starch layers, apparently on the expense of the gradually disappearing starch grains.The development of the pyrenoid was not directly affected by the colchicine treatment but 20 h later the undivided cells, obviously due to their polyploid and polynucleate state, contained several pyrenoids which frequently were combined in clusters and surrounded by several starch platelets.A redundant development of the endoplasmic reticulum was found if the cells were exposed to colchicine during the time of nuclear divisions. The number of Golgi bodies was increased per cell and per nucleus in the undivided, colchicine treated cells.