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.     

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．     

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.     

Immunogold Localization of Ribulose-1,5-Bisphosphate Carboxylase with Reference to Pyrenoid Morphology in Chloroplasts of Synchronized Euglena gracilis Cells

Euglena gracilis strain (Z) cells were synchronized under photoautotrophic conditions using a 14 hour light:10 hour dark regimen. The cells grew during the light period (growth phase) and divided during the following 10 hour period either in the dark or in the light (division phase). Changes in morphology of the pyrenoid and in the distribution of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) within the chloroplasts were followed by immunoelectron microscopy during the growth and division phases of Euglena cells. Epon-embedded sections were labeled with an antibody to the holoenzyme followed by protein A-gold. The immunoreactive proteins were concentrated in the pyrenoid, and less densely distributed in the stroma during the growth phase. During the division phase, the pyrenoid could not be detected and the gold particles were dispersed throughout the stroma. Toward the end of the division phase, the pyrenoid began to form in the center of a chloroplast, and the immunoreactive proteins started to concentrate over that rudimentary pyrenoid. During the growth phase, small areas rich in gold particles, called `satellite pyrenoid,' were observed, in addition to the main pyrenoid. From a comparison of photosynthetic CO₂-fixation with the total carboxylase activity of Rubisco extracted from Euglena cells in the growth phase, it is suggested that the carboxylase in the pyrenoid functions in CO₂-fixation in photosynthesis.     

Immunolocalization and distribution of form ii rubisco in the pyrenoid and chloroplast stroma of amphidinium carterae and form i rubisco in the symbiont-derived plastids of peridinium foliaceum (dinophyceae)

Chloroplasts of peridinin-containing dinoflagellates have recently been shown to contain Form II Rubisco, which consists of large subunits only and is coded by nuclear genes. We have used immunoelectron microscopy to determine the distribution of Form II and Form I Rubisco in dinoflagellates. In sections of Amphidinium carterae Hulburt, the pyrenoid was intensely labeled and the rest of the chloroplast moderately labeled by antisera to Form II Rubisco from the purple non-sulfur bacterium Rhodospirillum rubrum and the symbiotic dinoflagellate Symbiodinium sp. No labeling was observed when sections were exposed to antiserum against Form I Rubisco of the haptophyte alga Isochrysis galbana. In contrast, cell sections of the dinoflagellate Peridinium foliaceum (Stein) Biecheler, whose chloroplasts belong to a diatom endosymbiont, showed no labeling with the two antisera against Form II Rubisco, but heavy pyrenoid labeling was present after treatment with antiserum against Form I Rubisco of I. galbana. The same immunolabeling results were obtained with the free-living diatom Phaeodactylum tricornutum Bohlin. Volumetric analysis of the distribution of Form II Rubisco in the chloroplast of A. carterae showed that, in cells grown under moderate photon irradiance, 72.9% of the plastid's Rubisco was localized in the pyrenoid, whereas in cells grown under low irradiance only 37.0% of the Rubisco was found in the pyrenoid. This light-induced concentration of Rubisco in the pyrenoid suggests that a CO₂–concentrating mechanism may elevate CO₂ within the pyrenoid, favoring the efficient fixation of CO₂ by pyrenoid Rubisco.     

The CO2-concentrating mechanism in a starchless mutant of the green unicellular alga Chlorella pyrenoidosa

The CO₂-concentrating mechanism (CCM) was induced in the green unicellular alga Chlorella when cells were transferred from high (5% CO₂) to low (0.03%) CO₂ concentrations. The induction of the CCM correlated with the formation of a starch sheath specifically around the pyrenoid in the chloroplast. With the aim of clarifying whether the starch sheath was involved in the operation of the CCM, we isolated and physiologically characterized a starchless mutant of Chlorella pyrenoidosa, designated as IAA-36. The mutant strain grew as vigorously as the wild type under high and low CO₂ concentrations, continuous light and a 12 h light/12 h dark photoperiod. The CO₂ requirement for half-maximal rates of photosynthesis [K_0.5(CO₂)] decreased from 40 μM to 2–3 μM of CO₂ when both wild type and mutant were switched from high to low CO₂. The high affinity for inorganic carbon indicates that the IAA-36 mutant is able to induce a fully active CCM. Since the mutant does not have the pyrenoid starch sheath, we conclude that the sheath is not involved in the operation of the CCM in Chlorella cells.     

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.     

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.     

The Intracellular Localization of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase in Chlamydomonas reinhardtii

The pyrenoid is a proteinaceous structure found in the chloroplast of most unicellular algae. Various studies indicate that ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is present in the pyrenoid, although the fraction of Rubisco localized there remains controversial. Estimates of the amount of Rubisco in the pyrenoid of Chlamydomonas reinhardtii range from 5% to nearly 100%. Using immunolocalization, the amount of Rubisco localized to the pyrenoid or to the chloroplast stroma was estimated for C. reinhardtii cells grown under different conditions. It was observed that the amount of Rubisco in the pyrenoid varied with growth condition; about 40% was in the pyrenoid when the cells were grown under elevated CO₂ and about 90% with ambient CO₂. In addition, it is likely that pyrenoidal Rubisco is active in CO₂ fixation because in vitro activity measurements showed that most of the Rubisco must be active to account for CO₂-fixation rates observed in whole cells. These results are consistent with the idea that the pyrenoid is the site of CO₂ fixation in C. reinhardtii and other unicellular algae containing CO₂-concentrating mechanisms.     

CO2‐fixing liquid droplets: Towards a dissection of the microalgal pyrenoid

CO₂ enters the biosphere via the slow, oxygen‐sensitive carboxylase, Rubisco. To compensate, most microalgae saturate Rubisco with its substrate gas through a carbon dioxide concentrating mechanism. This strategy frequently involves compartmentalization of the enzyme in the pyrenoid, a non‐membrane enclosed compartment of the chloroplast stroma. Recently, tremendous advances have been achieved concerning the structure, physical properties, composition and in vitro reconstitution of the pyrenoid matrix from the green alga Chlamydomonas reinhardtii. The discovery of the intrinsically disordered multivalent Rubisco linker protein EPYC1 provided a biochemical framework to explain the subsequent finding that the pyrenoid resembles a liquid droplet in vivo. Reconstitution of the corresponding liquid‐liquid phase separation using pure Rubisco and EPYC1 allowed a detailed characterization of this process. Finally, a large high‐quality dataset of pyrenoidal protein‐protein interactions inclusive of spatial information provides ample substrate for rapid further functional dissection of the pyrenoid. Integrating and extending recent advances will inform synthetic biology efforts towards enhancing plant photosynthesis as well as contribute a versatile model towards experimentally dissecting the biochemistry of enzyme‐containing membraneless organelles.     

In Situ Association of Calvin Cycle Enzymes,Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Activase,Ferredoxin-NADP+ Reductase,and Nitrite Reductase with Thylakoid and Pyrenoid Membranes of Chlamydomonas reinhardtii Chloroplasts as Revealed by Immunoelectron Microscopy

The in situ localization of the chloroplast enzymes ribulose-1,5-bisphosphate carboxylase (Rubisco), Rubisco activase, ribose-5-phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, aldolase, nitrite reductase, ferredoxin-NADP+ reductase, and H+-ATP synthase was studied by immunoelectron microscopy in Chlamydomonas reinhardtii. Immunogold labeling revealed that, despite Rubisco in the pyrenoid matrix, Calvin cycle enzymes, Rubisco activase, nitrite reductase, ferredoxin-NADP+ reductase, and H+-ATP synthase are associated predominantly with chloroplast thylakoid membranes and the inner surface of the pyrenoid membrane. This is in accord with previous enzyme localization studies in higher plants (K.H. Suss, C. Arkona, R. Manteuffel, K. Adler [1993] Proc Natl Acad Sci USA 90: 5514-5518). Pyrenoid tubules do not contain these enzymes. The pyrenoid matrix consists of Rubisco but is devoid of the other photosynthetic enzymes investigated. Evidence for the occurrence of two Rubisco forms differing in their spatial localization has also been obtained: Rubisco form I appears to be membrane associated like other Calvin cycle components, whereas Rubisco form II is confined to the pyrenoid matrix. It is proposed that enzyme form I represents an active Rubisco when assembled into Calvin cycle enzyme complexes, whereas Rubisco form II may be part of a CO2-concentrating mechanism. Pyrenoidal Calvin cycle complexes are thought to be highly active in CO2 fixation and important for the synthesis of starch around the pyrenoid.     

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.     

The induction of the CO2 concentrating mechanism in a starch-less mutant of Chlamydomonas reinhardtii

In Chlamydomonas reinhardtii the formation of a starch sheath surrounding the pyrenoid is observed when cells grown under high CO₂ (5% CO₂ in air) are transferred to low CO₂ (0.03%) conditions. Formation of the starch sheath occurs coincidentally with induction of the CO₂ concentrating mechanism and with de novo synthesis of 5 polypeptides with molecular masses of 21, 36, 37, 42–44 kDa. We studied the effect of CO₂ concentrations on photosynthesis, ultrastructure and protein synthesis in Chlamydomonas reinhardtii cw-15 (wild phenotype for photosynthesis) and in the starch-less mutant BAFJ -6, with the aim to clarify the role of the pyrenoid starch sheath in the operation of the CO₂ concentrating mechanism and whether these low CO₂-inducible polypeptides are involved in the formation of starch sheath. When wild type and starch-less mutant cells were transferred from high to low CO₂, the CO₂ requirement for half-maximal rates of photosynthesis decreased from 40 μM to 2 μM CO₂. ³⁵SO₄^2- labeling analyses showed that the starch-less mutant induced the 5 low CO₂-inducible polypeptides. These observations suggest that the starch-less mutant was able to induce a fully active CO₂ concentrating mechanism. Since the starch-less mutant did not form a pyrenoid starch sheath, we suggest that the starch sheath is not involved in the operation of the CO₂ concentrating mechanism and that none of these 5 low CO₂-inducible proteins is involved in the formation of the starch sheath in Chlamydomonas .     

Variation in Net Photosynthesis, Rubisco Activity and Chloroplast Ultrastructure among Somatic Hybrids of Solanum tuberosum and S. brevidens

The effect of ploidy, parental chloroplast type and parentalnuclear genome dosage on net photosynthesis, Rubisco activityand chloroplast ultrastructure was studied among somatic hybridsof diploid S. brevidens and dihaploid S. tuberosum. An increasein nuclear ploidy resulted in an increase in net photosynthesisand specific leaf weight. There were no significant differencesin net photosynthesis or Rubisco activity between the hybridshaving different parental chloroplast type. Examination of thehexaploid hybrids indicated that Rubisco activity was affectedby nuclear-organelle genome incompatibility, the most affectedcombination being tuberosum chloroplast type with brevidensnuclear genome. Examination of chloroplast ultrastructure revealedwide variation in the size of chloroplasts, starch granules,plastoglobuli and in grana stacking among the hybrids and betweenfusion parents. Key words: Somatic hybrids, Solanum, net photosynthesis, Rubisco, chloroplast ultrastructure     

Degradation of Ribulose-1, 5-Bisphosphate Carboxylase/Oxygenase in Wheat Leaves During Dark-induced Senescence

The degradation of Ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) in wheat (Triticum aestivum L. cv. Yangmai 158) leaves during dark-induced senescence was studied. An in vivo degradation product of Rubisco large subunit (LSU) with molecular weight of 50 kD was detected by SDS-PAGE and immunoblotting with antibody against tobacco Rubisco. This fragment could also be detected in natural senescence. The result also suggested that the Rubisco holoenzyme had not dissociated when LSU hydrolyzed from 53 kD to 50 kD. And LSU could be fragmented to 50 kD at 30-35 ℃ and at pH 7.5 in crude enzyme extracts of wheat leaves dark-induced for 48 h, which suggested that maybe LSU was degraded to 50 kD by an unknown protease in chloroplast.     

Functional Hybrid Rubisco Enzymes with Plant Small Subunits and Algal Large Subunits: ENGINEERED rbcS cDNA FOR EXPRESSION IN CHLAMYDOMONAS*?

There has been much interest in the chloroplast-encoded large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) as a target for engineering an increase in net CO₂ fixation in photosynthesis. Improvements in the enzyme would lead to an increase in the production of food, fiber, and renewable energy. Although the large subunit contains the active site, a family of rbcS nuclear genes encodes the Rubisco small subunits, which can also influence the carboxylation catalytic efficiency and CO₂/O₂ specificity of the enzyme. To further define the role of the small subunit in Rubisco function, small subunits from spinach, Arabidopsis, and sunflower were assembled with algal large subunits by transformation of a Chlamydomonas reinhardtii mutant that lacks the rbcS gene family. Foreign rbcS cDNAs were successfully expressed in Chlamydomonas by fusing them to a Chlamydomonas rbcS transit peptide sequence engineered to contain rbcS introns. Although plant Rubisco generally has greater CO₂/O₂ specificity but a lower carboxylation V_max than Chlamydomonas Rubisco, the hybrid enzymes have 3–11% increases in CO₂/O₂ specificity and retain near normal V_max values. Thus, small subunits may make a significant contribution to the overall catalytic performance of Rubisco. Despite having normal amounts of catalytically proficient Rubisco, the hybrid mutant strains display reduced levels of photosynthetic growth and lack chloroplast pyrenoids. It appears that small subunits contain the structural elements responsible for targeting Rubisco to the algal pyrenoid, which is the site where CO₂ is concentrated for optimal photosynthesis.     

NEW ULTRASTRUCTURAL ASPECTS OF PYRENOIDS OF THE LICHEN PHOTOBIONT TREBOUXZA (MICROTHAMNIALES,CHLOROPHYTA)1

The pyrenoid structure of Trebouxia, a photobiont of two lichen species, Umbilicaria cinereorufescens (Schaer.) Frey and Parmelia sulcata Taylor, was investigated. In both lichen species, the pyrenoid of the photobiont exhibited straight, unbranched, long or short tubules. In the first lichen species, multiple pyrenoids were observed occasionally, while in the second one, homogeneous masses, called protein bodies, appeared between the thylakoids. These protein bodies were previously observed in some other species of the family Umbilicariaceae. Serial sections from single pyrenoids showed that tubules of the Impressa-type pyrenoid were closely associated with pyrenoglobuli. The three-dimensional reconstruction of a complete chloroplast of a P. sulcata algal cell showed that the protein bodies were spatially separate structures. Immunolocalization techniques to detect the presence of ribulose-bisphosphate carboxylase (Rubisco) in the chloroplast showed that this enzyme was present primarily in the pyrenoid matrix. When protein bodies were present in the chloroplast, Rubisco appeared to be localized in these structures. The presence of pyrenoid satellites and protein bodies with reactivity to anti-Rubisco may be related to the nutritional conditions of the thalli.     

Chloroplast structure and starch grain production as phylogenetic indicators in the lower Rhodophyceae

The position of starch grain production, the shape of the starch grains and the depth to which the pyrenoid is embedded in the chloroplast are used as indicators of evolution in the lower Rhodophyceae. A cell with cytoplasmic allantoid starch grains encasing the pyrenoid and no thylakoids between the chloroplast envelope and the pyrenoid is considered to be evolutionarily primitive. A cell with oval starch grains not associated with the pyrenoid and with a pyrenoid deeply embedded in the chloroplast is thought to be evolutionarily advanced. A polyphyletic origin of the Porphyridiales is discussed.