衣藻有性生殖的分子机制

衣藻作为分子生物学研究的模式材料,被广泛用于植物光合作用、鞭毛组装与功能、细胞周期与节律、细胞信号传导与光感受、细胞识别等重要生物学过程的研究,而且衣藻有性生殖的分子机制与人类某些疾病的发生机制存在联系.该文对国内外近年来有关莱茵衣藻在有性生殖过程中凝集素的动态分布,包括鞭毛粘连、补充、传递、脱粘连、凝集素合成的正调节,以及与性凝集素行为有关的基因研究进展进行综述,以阐明衣藻有性生殖的分子机制,为人类的疾病研究提供参考.     

Chlamydomonas reinhardtii: biological rationale for genomics

Chlamydomonas reinhardtii has been the subject of genetic, biochemical, cytological, and molecular analyses for over 50 years. It is an ideal model system for the study of flagella and basal bodies as well as the study of photosynthesis and chloroplast biogenesis, cell-cell recognition and fusion, phototaxis, and secretion. It is clear that many of the genes identified in Chlamydomonas have homologs in land plants as well as animals. Thus, a genomic approach in Chlamydomonas will provide another important avenue for the understanding of important biological processes.     

Functional genomics of eukaryotic photosynthesis using insertional mutagenesis of Chlamydomonas reinhardtii

The unicellular green alga Chlamydomonas reinhardtii is a widely used model organism for studies of oxygenic photosynthesis in eukaryotes. Here we describe the development of a resource for functional genomics of photosynthesis using insertional mutagenesis of the Chlamydomonas nuclear genome. Chlamydomonas cells were transformed with either of two plasmids conferring zeocin resistance, and insertional mutants were selected in the dark on acetate-containing medium to recover light-sensitive and nonphotosynthetic mutants. The population of insertional mutants was subjected to a battery of primary and secondary phenotypic screens to identify photosynthesis-related mutants that were pigment deficient, light sensitive, nonphotosynthetic, or hypersensitive to reactive oxygen species. Approximately 9% of the insertional mutants exhibited 1 or more of these phenotypes. Molecular analysis showed that each mutant line contains an average of 1.4 insertions, and genetic analysis indicated that approximately 50% of the mutations are tagged by the transforming DNA. Flanking DNA was isolated from the mutants, and sequence data for the insertion sites in 50 mutants are presented and discussed.     

Chlamydomonas phototaxis

Chlamydomonas has long been a favourite organism for genetic and biochemical studies of flagellar motility and assembly, photosynthesis, and organelle genomes. With the recent development of procedures for the efficient transformation of its nuclear genome, Chlamydomonas has become accessible to a wide range of molecular genetic approaches, including gene tagging by insertional mutagenesis and cloning by complementation. The availability of these powerful techniques is stimulating interest in Chlamydomonas as a model system for research in areas where it previously has not been widely exploited. One such area that holds particular promise is phototransduction and the behavioural response to light.     

模式生物衣藻及其研究进展

单细胞衣藻（Chlamydomonas）由于其生活周期简单,培养方法简便,易于分离得到系列的突变体,并已建立了分子遗传学研究技术与遗传分析系统,成为植物光合作用、鞭毛组装与功能、细胞周期及节律、细胞信号传导与光感受、细胞识别等重要生物学过程研究的模式生物体。本文对模式生物衣藻及其相关生物学途径的研究进展作一综述。 Abstract:The unicellular alga Chlamydomonas offers a simple life cycle,easy culture and isolation of series of mutants,established the techniques and tool kit for molecular genetics and genetics analysis.It is now becoming the model organism for studies on photosynthesis in plant,flagellar assembly and function,cell cycle and circadian rhythms,signal transduction,light perception and cell recognition.It is summarized the progress of study on Chlamydomonas as a model organism in this paper.     

Beyond complementation. Map-based cloning in Chlamydomonas reinhardtii

Chlamydomonas reinhardtii is an excellent model system for plant biologists because of its ease of manipulation, facile genetics, and the ability to transform the nuclear, chloroplast, and mitochondrial genomes. Numerous forward genetics studies have been performed in Chlamydomonas, in many cases to elucidate the regulation of photosynthesis. One of the resultant challenges is moving from mutant phenotype to the gene mutation causing that phenotype. To date, complementation has been the primary method for gene cloning, but this is impractical in several situations, for example, when the complemented strain cannot be readily selected or in the case of recessive suppressors that restore photosynthesis. New tools, including a molecular map consisting of 506 markers and an 8X-draft nuclear genome sequence, are now available, making map-based cloning increasingly feasible. Here we discuss advances in map-based cloning developed using the strains mcd4 and mcd5, which carry recessive nuclear suppressors restoring photosynthesis to chloroplast mutants. Tools that have not been previously applied to Chlamydomonas, such as bulked segregant analysis and marker duplexing, are being implemented to increase the speed at which one can go from mutant phenotype to gene. In addition to assessing and applying current resources, we outline anticipated future developments in map-based cloning in the context of the newly extended Chlamydomonas genome initiative.     

The slow S to M rise of chlorophyll a fluorescence reflects transition from state 2 to state 1 in the green alga Chlamydomonas reinhardtii

Photosynthesis Research - The green alga Chlamydomonas (C.) reinhardtii is a model organism for photosynthesis research. State transitions regulate redistribution of excitation energy between...     

Analytical approaches to photobiological hydrogen production in unicellular green algae

Photosynthesis Research - Several species of unicellular green algae, such as the model green microalga Chlamydomonas reinhardtii, can operate under either aerobic photosynthesis or anaerobic...     

Genome analysis of Chlamydomonas reinhardtii reveals the existence of multiple, compartmentalized iron-sulfur protein assembly machineries of different evolutionary origins

The unicellular green alga Chlamydomonas reinhardtii is used extensively as a model to study eukaryotic photosynthesis, flagellar functions, and more recently the production of hydrogen as biofuel. Two of these processes, photosynthesis and hydrogen production, are highly dependent on iron-sulfur (Fe-S) enzymes. To understand how Fe-S proteins are assembled in Chlamydomonas, we have analyzed its recently sequenced genome for orthologs of genes involved in Fe-S cluster assembly. We found a total of 32 open reading frames, most single copies, that are thought to constitute a mitochondrial assembly pathway, mitochondrial export machinery, a cytosolic assembly pathway, and components for Fe-S cluster assembly in the chloroplast. The chloroplast proteins are also expected to play a role in the assembly of the H-cluster in [FeFe]-hydrogenases, together with the recently identified HydEF and HydG proteins. Comparison with the higher plant model Arabidopsis indicated a strong degree of conservation of Fe-S cofactor assembly pathways in the green lineage, the pathways being derived from different origins during the evolution of the photosynthetic eukaryote. As a haploid, unicellular organism with available forward and reverse genetic tools, Chlamydomonas provides an excellent model system to study Fe-S cluster assembly and its regulation in photosynthetic eukaryotes.     

Between a rock and a hard place: trace element nutrition in Chlamydomonas

Photosynthetic organisms are among the earliest life forms on earth and their biochemistry is strictly dependent on a wide range of inorganic nutrients owing to the use of metal cofactor-dependent enzymes in photosynthesis, respiration, inorganic nitrogen and sulfur assimilation. Chlamydomonas reinhardtii is a photosynthetic eukaryotic model organism for the study of trace metal homeostasis. Chlamydomonas spp. are widely distributed and can be found in soil, glaciers, acid mines and sewage ponds, suggesting that the genus has significant capacity for acclimation to micronutrient availability. Analysis of the draft genome indicates that metal homeostasis mechanisms in Chlamydomonas represent a blend of mechanisms operating in animals, plants and microbes. A combination of classical genetics, differential expression and genomic analysis has led to the identification of homologues of components known to operate in fungi and animals (e.g., Fox1, Ftr1, Fre1, Fer1, Ctr1/2) as well as novel molecules involved in copper and iron nutrition (Crr1, Fea1/2). Besides activating iron assimilation pathways, iron-deficient Chlamydomonas cells re-adjust metabolism by reducing light delivery to photosystem I (to avoid photo-oxidative damage resulting from compromised FeS clusters) and by modifying the ferredoxin profile (perhaps to accommodate preferential allocation of reducing equivalents). Up-regulation of a MnSOD isoform may compensate for loss of FeSOD. Ferritin could function to buffer the iron released from programmed degradation of iron-containing enzymes in the chloroplast. Some metabolic adjustments are made in anticipation of deficiency while others occur only with sustained or severe deficiency. Copper-deficient Chlamydomonas cells induce a copper assimilation pathway consisting of a cell surface reductase and a Cu(+) transporter (presumed CTR homologue). There are metabolic adaptations in addition: the synthesis of "back-up" enzymes for plastocyanin in photosynthesis and the ferroxidase in iron assimilation plus activation of alternative oxidase to handle the electron "overflow" resulting from reduced cytochrome oxidase function. Oxygen-dependent enzymes in the tetrapyrrole pathway (coproporphyrinogen oxidase and aerobic oxidative cyclase) are also increased in expression and activity by as much as 10-fold but the connection between copper nutrition and tetrapyrroles is not understood. The copper-deficiency responses are mediated by copper response elements that are defined by a GTAC core sequence and a novel metalloregulator, Crr1, which uses a zinc-dependent SBP domain to bind to the CuRE. The Chlamydomonas model is ideal for future investigation of nutritional manganese deficiency and selenoenzyme function. It is also suited for studies of trace nutrient interactions, nutrition-dependent metabolic changes, the relationship between photo-oxidative stress and metal homeostasis, and the important questions of differential allocation of limiting metal nutrients (e.g., to respiration vs. photosynthesis).     

Mechanisms of response to salinity in halotolerant microalgae

Summary A limited number of organic solutes are used by microalgae to adjust their internal osmotic pressure in response to changing external salinities. Glycerol and proline are used by the most extremely halotolerant algae. Only glycerol allows growth at salinities approaching saturation. In addition to organic osmoregulatory solutes, inorganic ions also play an important role in osmoregulation. The ability of microalgae to maintain intracellular ions at levels compatible with metabolic functions may set upper limits for their salt tolerance. Requirements for NaCl in the external medium for nutrient transport may define the lower salinity limits for growth observed for some euryhaline algae.Osmotic upshocks generally cause severe temporary inhibition of photosynthesis in euryhaline microalgae. Extensive osmotic downshocks have little effect on photosynthesis in microalgae with strong cell walls, while wall-less species appear to be more sensitive. Rapid glycerol synthesis takes place in response to increased external salinity inChlamydomonas pulsatilla both in light and dark. Starch supplies carbon for glycerol synthesis in the dark and also during the initial periods of inhibition of photosynthesis in the light. Turnover of osmoregulatory solutes such as glycerol and isofloridoside may be an important aspect of the osmoregulatory mechanism.At salinities beyond the growth limit for the green flagellateChlamydomonas pulsatilla, resting spores are formed that enable this alga to survive extreme salinities.     

Mutants of Chlamydomonas: tools to study thylakoid membrane structure, function and biogenesis.

The unicellular green alga Chlamydomonas reinhardtii is a model system for the study of photosynthesis and chloroplast biogenesis. C. reinhardtii has a photosynthesis apparatus similar to that of higher plants and it grows at rapid rate (generation time about 8 h). It is a facultative phototroph, which allows the isolation of mutants unable to perform photosynthesis and its sexual cycle allows a variety of genetic studies. Transformation of the nucleus and chloroplast genomes is easily performed. Gene transformation occurs mainly by homologous recombination in the chloroplast and heterologous recombination in the nucleus. Mutants are precious tools for studies of thylakoid membrane structure, photosynthetic function and assembly. Photosynthesis mutants affected in the biogenesis of a subunit of a protein complex usually lack the entire complex; this pleiotropic effect has been used in the identification of the other subunits, in the attribution of spectroscopic signals and also as a 'genetic cleaning' process which facilitates both protein complex purification, absorption spectroscopy studies or freeze-fracture analysis. The cytochrome b6f complex is not required for the growth of C. reinhardtii, unlike the case of photosynthetic prokaryotes in which the cytochrome complex is also part of the respiratory chain, and can be uniquely studied in Chlamydomonas by genetic approaches. We describe in greater detail the use of Chlamydomonas mutants in the study of this complex.     

Analysis of Chlamydomonas reinhardtii genome structure using large-scale sequencing of regions on linkage groups I and III

Chlamydomonas reinhardtii is a unicellular green alga that has been used as a model organism for the study of flagella and basal bodies as well as photosynthesis. This report analyzes finished genomic DNA sequence for 0.5% of the nuclear genome. We have used three gene prediction programs as well as EST and protein homology data to estimate the total number of genes in Chlamydomonas to be between 12,000 and 16,400. Chlamydomonas appears to have many more genes than any other unicellular organism sequenced to date. Twenty-seven percent of the predicted genes have significant identity to both ESTs and to known proteins in other organisms, 32% of the predicted genes have significant identity to ESTs alone, and 14% have significant similarity to known proteins in other organisms. For gene prediction in Chlamydomonas, GreenGenie appeared to have the highest sensitivity and specificity at the exon level, scoring 71% and 82%. respectively. Two new alternative splicing events were predicted by aligning Chlamydomonas ESTs to the genomic sequence. Finally recombination differs between the two sequenced contigs. The 350-Kb of the Linkage group III contig is devoid of recombination, while the Linkage group I contig is 30 map units long over 33-kb.     

Detecting mutants that have impaired photosynthesis by their increased level of fluorescence

A technique is described for detecting mutants of Chlamydomonas reinhardi that have impaired photosynthesis. It is based on the fact that when photosynthetic electron transport is blocked there may be an increase in the level of fluorescence similar to that obtained in the presence of an inhibitor of photosynthetic electron transport such as dichlorophenyldimethylurea.     

Novel metabolism in Chlamydomonas through the lens of genomics

Chlamydomonas has traditionally been exploited as an organism that is associated with sophisticated physiological, genetic and molecular analyses, all of which have been used to elucidate several biological processes, especially photosynthesis and flagella function and assembly. Recently, the genomics of Chlamydomonas has been combined with other technologies to unveil new aspects of metabolism, including inorganic carbon utilization, anaerobic fermentation, the suite and functions of selenoproteins, and the regulation of vitamin biosynthesis. These initial findings represent the first glimpse through a genomic window onto the highly complex metabolisms that characterize a unicellular, photosynthetic eukaryote that has maintained both plant-like and animal-like characteristics over evolutionary time.     

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 .     

Novel insights into the function of LHCSR3 in Chlamydomonas reinhardtii

Light is essential for photosynthesis but excess light is hazardous as it may lead to the formation of reactive oxygen species. Photosynthetic organisms struggle to optimize light utilization and photosynthesis while minimizing photo-oxidative damage. Hereby light to heat dissipation via specialized proteins is a potent mechanism to acclimate toward excess light. In the green alga Chlamydomonas reinhardtii the expression of an ancient light-harvesting protein LHCSR3 enables cells to dissipate harmful excess energy. Herein we summarize newest insights into the function of LHCSR3 from C. reinhardtii.     

Carotenoid deficiency triggers autophagy in the model green alga Chlamydomonas reinhardtii

All aerobic organisms have developed sophisticated mechanisms to prevent, detect and respond to cell damage caused by the unavoidable production of reactive oxygen species (ROS). Plants and algae are able to synthesize specific pigments in the chloroplast called carotenoids to prevent photo-oxidative damage caused by highly reactive by-products of photosynthesis. In this study we used the unicellular green alga Chlamydomonas reinhardtii to demonstrate that defects in carotenoid biosynthesis lead to the activation of autophagy, a membrane-trafficking process that participates in the recycling and degradation of damaged or toxic cellular components. Carotenoid depletion caused by either the mutation of phytoene synthase or the inhibition of phytoene desaturase by the herbicide norflurazon, resulted in a strong induction of autophagy. We found that high light transiently activates autophagy in wild-type Chlamydomonas cells as part of an adaptation response to this stress. Our results showed that a Chlamydomonas mutant defective in the synthesis of specific carotenoids that accumulate during high light stress exhibits constitutive autophagy. Moreover, inhibition of the ROS-generating NADPH oxidase partially reduced the autophagy induction associated to carotenoid deficiency, which revealed a link between photo-oxidative damage, ROS accumulation and autophagy activation in Chlamydomonas cells with a reduced carotenoid content.