Induction and characterization of mitochondrial DNA mutants in Chlamydomonas reinhardtii

In addition to lethal minute colony mutations which correspond to loss of mitochondrial DNA, acriflavin induces in Chlamydomonas reinhardtii a low percentage of cells that grow in the light but do not divide under heterotrophic conditions. Two such obligate photoautotrophic mutants were shown to lack the cyanide-sensitive cytochrome pathway of the respiration and to have a reduced cytochrome c oxidase activity. In crosses to wild type, the mutations are transmitted almost exclusively from the mating type minus parent. A same pattern of inheritance is seen for the mitochondrial DNA in crosses between the two interfertile species C. reinhardtii and Chlamydomonas smithii. Both mutants have a deletion in the region of the mitochondrial DNA containing the apocytochrome b gene and possibly the unidentified URFx gene.     

Molecular remodeling of photosystem II during state transitions in Chlamydomonas reinhardtii

State transitions, or the redistribution of light-harvesting complex II (LHCII) proteins between photosystem I (PSI) and photosystem II (PSII), balance the light-harvesting capacity of the two photosystems to optimize the efficiency of photosynthesis. Studies on the migration of LHCII proteins have focused primarily on their reassociation with PSI, but the molecular details on their dissociation from PSII have not been clear. Here, we compare the polypeptide composition, supramolecular organization, and phosphorylation of PSII complexes under PSI- and PSII-favoring conditions (State 1 and State 2, respectively). Three PSII fractions, a PSII core complex, a PSII supercomplex, and a multimer of PSII supercomplex or PSII megacomplex, were obtained from a transformant of the green alga Chlamydomonas reinhardtii carrying a His-tagged CP47. Gel filtration and single particles on electron micrographs showed that the megacomplex was predominant in State 1, whereas the core complex was predominant in State 2, indicating that LHCIIs are dissociated from PSII upon state transition. Moreover, in State 2, strongly phosphorylated LHCII type I was found in the supercomplex but not in the megacomplex. Phosphorylated minor LHCIIs (CP26 and CP29) were found only in the unbound form. The PSII subunits were most phosphorylated in the core complex. Based on these observations, we propose a model for PSII remodeling during state transitions, which involves division of the megacomplex into supercomplexes, triggered by phosphorylation of LHCII type I, followed by LHCII undocking from the supercomplex, triggered by phosphorylation of minor LHCIIs and PSII core subunits.     

Thylakoid membrane dynamics and state transitions in Chlamydomonas reinhardtii under elevated temperature

Photosynthesis Research - Moderately elevated temperatures can induce state transitions in higher plants by phosphorylation of light-harvesting complex II (LHCII). In this study, we exposed...     

New arginine-requiring mutants in Chlamydomonas reinhardtii

Twelve arginine-requiring mutants of the unicellular green alga Chlamydomonas reinhardtii previously isolated in our laboratory were investigated to find new blocks in the biosynthetic pathway of arginine. In addition to the already described mutants lacking acetylglutamyl phosphate reductase (arg 1), ornithine carbamoyltransferase (arg4) and argininosuccinate lyase (arg7), three new types of mutants were found lacking acetylornithine aminotransferase (arg9-1, arg9-2), acetylornithine glutamate transacetylase (arg10) and argininosuccinate synthetase (arg8-1, arg8-2, arg8-3) respectively. The genetic analysis of these new mutants showed that arg9 and arg8 are unlinked to the other arginine markers and that arg10 probably carries a chromosomal mutation inducing a very high lethality of meiotic products.Abbreviations WT wild-type - mt mating-type - SP spore plating - ZP zygote plating     

Eyespot-assembly mutants in Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii is a single-celled green alga that phototaxes toward light by means of a light-sensitive organelle, the eyespot. The eyespot is composed of photoreceptor and Ca(++)-channel signal transduction components in the plasma membrane of the cell and reflective carotenoid pigment layers in an underlying region of the large chloroplast. To identify components important for the positioning and assembly of a functional eyespot, a large collection of nonphototactic mutants was screened for those with aberrant pigment spots. Four loci were identified. eye2 and eye3 mutants have no pigmented eyespots. min1 mutants have smaller than wild-type eyespots. mlt1(ptx4) mutants have multiple eyespots. The MIN1, MLT1(PTX4), and EYE2 loci are closely linked to each other; EYE3 is unlinked to the other three loci. The eye2 and eye3 mutants are epistatic to min1 and mlt1 mutations; all double mutants are eyeless. min1 mlt1 double mutants have a synthetic phenotype; they are eyeless or have very small, misplaced eyespots. Ultrastructural studies revealed that the min1 mutants are defective in the physical connection between the plasma membrane and the chloroplast envelope membranes in the region of the pigment granules. Characterization of these four loci will provide a beginning for the understanding of eyespot assembly and localization in the cell.     

Isolation of state transition mutants of Chlamydomonas reinhardtii by fluorescence video imaging

Oxygenic photosynthetic organisms adapt to varying light conditions by changing the distribution of light energy between Photosystem II (PS II) and photosystem I (PS I) during so-called state transitions. To identify the genes involved in this process, we have exploited a simple chlorophyll fluorescence video-imaging technique to screen a library of nuclear mutants of Chlamydomonas reinhardtii for colonies grown on agar plates that are disturbed in their ability to regulate light energy distribution between PS I and PS II. Subsequent modulated fluorescence measurements at room temperature and 77 K fluorescence emission spectra confirmed that 5 mutants (0.025% of total number screened) were defective in state transitions. [³²P]orthophosphate phosphorylation experiments in vivo revealed that in one of these mutants, designated stm1, the level of LHC II polypeptide phosphorylation was drastically reduced compared with wild type. Despite WT levels of PS I and PS II, stm1 grew photoautotrophically at reduced rates, compared with WT especially under low light conditions, which is consistent with an important physiological role for state transitions. Our results highlight the feasibility of video imaging in tandem with mutagenesis as a means of identifying the genes involved in controlling state transitions in eukaryotic photosynthetic organisms.     

Isolation and characterization of photoautotrophic mutants of Chlamydomonas reinhardtii deficient in state transition.

In photosynthetic cells of higher plants and algae, the distribution of light energy between photosystem I and photosystem II is controlled by light quality through a process called state transition. It involves a reorganization of the light-harvesting complex of photosystem II (LHCII) within the thylakoid membrane whereby light energy captured preferentially by photosystem II is redirected toward photosystem I or vice versa. State transition is correlated with the reversible phosphorylation of several LHCII proteins and requires the presence of functional cytochrome b(6)f complex. Most factors controlling state transition are still not identified. Here we describe the isolation of photoautotrophic mutants of the unicellular alga Chlamydomonas reinhardtii, which are deficient in state transition. Mutant stt7 is unable to undergo state transition and remains blocked in state I as assayed by fluorescence and photoacoustic measurements. Immunocytochemical studies indicate that the distribution of LHCII and of the cytochrome b(6)f complex between appressed and nonappressed thylakoid membranes does not change significantly during state transition in stt7, in contrast to the wild type. This mutant displays the same deficiency in LHCII phosphorylation as observed for mutants deficient in cytochrome b(6)f complex that are known to be unable to undergo state transition. The stt7 mutant grows photoautotrophically, although at a slower rate than wild type, and does not appear to be more sensitive to photoinactivation than the wild-type strain. Mutant stt3-4b is partially deficient in state transition but is still able to phosphorylate LHCII. Potential factors affected in these mutant strains and the function of state transition in C. reinhardtii are discussed.     

Flagellar morphology in stumpy-flagella mutants of Chlamydomonas reinhardtii

Sixteen new mutants of the biflagellate green alga Chlamydomonas reinhardtii with either stumpy-flagella or no flagella at all were examined by electron microscopy. Four of the mutants were found to carry short bulbous flagella containing amorphous electron-dense material which may represent unassembled flagellar protein. Basal bodies of normal ultrastructure were present in all mutants. Dikaryon dominance tests indicated that the stumpy mutations were recessive to wild-type in all cases tested. Stumpy mutations also conferred a measure of detergent resistance to Chlamydomonas, apparently by affecting the detergent-solubility of the flagellar membrane.     

Gravitaxis in Chlamydomonas reinhardtii studied with novel mutants

Many free-swimming unicellular organisms show negative gravitaxis, i.e. tend to swim upward, although their specific densities are higher than the medium density. To obtain clues to the mechanism of this behavior, we examined how a mutation in motility or behavior affects the gravitaxis in Chlamydomonas. A phototaxis mutant, ptx3, deficient in membrane excitability showed weakened gravitaxis, whereas another phototaxis mutant, ptx1, deficient in regulation of flagellar dominance displayed normal gravitaxis. Two mutants that swim backwards only, mbo1 and mbo2, did not show any clear gravitaxis. We also isolated two novel mutants deficient in gravitaxis, gtx1 and gtx2. These mutants displayed normal motility and physical characteristics of cell body as assessed by the behavior of anesthetized cells. However, these cells were found to have defects in physiological responses involving membrane excitation. These observations are consistent with the idea that the gravitaxis in Chlamydomonas involves a physiological signal transduction system, which is at least partially independent of the system used for phototaxis.     

Involvement of state transitions in the switch between linear and cyclic electron flow in Chlamydomonas reinhardtii

The energetic metabolism of photosynthetic organisms is profoundly influenced by state transitions and cyclic electron flow around photosystem I. The former involve a reversible redistribution of the light-harvesting antenna between photosystem I and photosystem II and optimize light energy utilization in photosynthesis whereas the latter process modulates the photosynthetic yield. We have used the wild-type and three mutant strains of the green alga Chlamydomonas reinhardtii—locked in state I (stt7), lacking the photosystem II outer antennae (bf4) or accumulating low amounts of cytochrome b₆f complex (A-AUU)—and measured electron flow though the cytochrome b₆f complex, oxygen evolution rates and fluorescence emission during state transitions. The results demonstrate that the transition from state 1 to state 2 induces a switch from linear to cyclic electron flow in this alga and reveal a strict cause–effect relationship between the redistribution of antenna complexes during state transitions and the onset of cyclic electron flow.     

A forward genetic screen identifies mutants deficient for mitochondrial complex I assembly in Chlamydomonas reinhardtii

Mitochondrial complex I is the largest multimeric enzyme of the respiratory chain. The lack of a model system with facile genetics has limited the molecular dissection of complex I assembly. Using Chlamydomonas reinhardtii as an experimental system to screen for complex I defects, we isolated, via forward genetics, amc1-7 nuclear mutants (for assembly of mitochondrial complex I) displaying reduced or no complex I activity. Blue native (BN)-PAGE and immunoblot analyses revealed that amc3 and amc4 accumulate reduced levels of the complex I holoenzyme (950 kDa) while all other amc mutants fail to accumulate a mature complex. In amc1, -2, -5-7, the detection of a 700 kDa subcomplex retaining NADH dehydrogenase activity indicates an arrest in the assembly process. Genetic analyses established that amc5 and amc7 are alleles of the same locus while amc1-4 and amc6 define distinct complementation groups. The locus defined by the amc5 and amc7 alleles corresponds to the NUOB10 gene, encoding PDSW, a subunit of the membrane arm of complex I. This is the first report of a forward genetic screen yielding the isolation of complex I mutants. This work illustrates the potential of using Chlamydomonas as a genetically tractable organism to decipher complex I manufacture.     

Accumulation of starch in Chlamydomonas reinhardtii flagellar mutants.

Paralyzed flagellar mutants pf-1, pf-2, pf-7, and pf-18 of the green alga Chlamydomonas reinhardtii (Dangeard) were shown to store a significantly greater amount of starch than the motile wild type 137c+. The increase in starch storage was significant relative to protein, chlorophyll, and cell number. Analysis of average cell size revealed that the paralyzed mutants were larger than the wild type. This increase in storage molecule accumulation supports an inverse relationship between chemical energy storage and energy utilization for biomechanical/motile cellular functions. Chlamydomonas reinhardtii provides a useful model for studies of the role of cytoskeletal activity in the energy relationship and balance of organisms.     

Cr6+胁迫对莱茵衣藻光合作用的影响

以莱茵衣藻(Chlamydomonas reinhardtii)为研究材料,采用氧电极和快速叶绿素a荧光诱导动力学方法研究了不同浓度和时间Cr6+处理对其光合作用的影响.结果表明:当Cr6+浓度大于40 μmol/L时,莱茵衣藻细胞数逐渐下降,而藻细胞变大;表观光合速率成为负值,呼吸作用随Cr6+处理浓度的增加先上升后下降至对照水平;莱茵衣藻有活性放氧复合体比例随Cr6+处理浓度的增加逐渐降低,80 μmol/L Cr6+处理3 d时已下降至13.72%;光合驱动力(DFABS)随Cr6+浓度增加逐步下降,并以DFφPo在DFABS的下降中的贡献最大.研究发现,重金属Cr6+胁迫显著影响莱茵衣藻的光合作用,而对呼吸作用则影响较小;Cr6+主要通过损伤供体侧的放氧复合体以及阻断QA至QB的电子传递而抑制光系统Ⅱ的功能;莱茵衣藻光系统Ⅱ对Cr6+处理比较敏感且存在着多个作用位点,并首先影响反应中心光能捕获效率,其次影响反应中心的活性,最后影响QA-之后的电子传递.     

The central role of the green alga Chlamydomonas reinhardtii in revealing the mechanism of state transitions

This review focuses on the essential role played by the green alga Chlamydomonas reinhardtii in revealing both the mechanism and the physiological consequences of state transitions. Two aspects are considered. The first is the role of the cytochrome b6f complex in regulating state transitions, in light of the recently obtained 3D structure. The second is the switch between linear and cyclic electron flow that follows state transitions in Chlamydomonas. Structural and dynamic elements that might be involved in such a switch, as well as its consequences on the energetic metabolism, are discussed.     

Insertional mutagenesis to isolate acetate-requiring mutants in Chlamydomonas reinhardtii

Abstract An arg 7 mutant of the green alga Chlamydomonas reinhardtii was transformed with pARG7.8, a plasmid bearing the wild-type ARG 7 gene. Out of 4100 arg⁺ transformants selected on an arginine-free medium supplemented with acetate, nine failed to grow on acetate-free medium (ac⁻ mutants). The results of the genetic and molecular analysis of several ac⁻ mutants are in agreement with the hypothesis that they originated from insertion of the incoming plasmid into the nuclear genome. These mutants should constitute valuable tools for isolating the corresponding wild-type genes after plasmid rescue into Escherichia coli .     

Form of Inorganic Carbon Utilized for Photosynthesis in Chlamydomonas reinhardtii1

The effect of carbonic anhydrase (CA) on time courses of photosynthetic¹⁴C incorporation in the presence of ¹⁴CO₂ or NaH¹⁴CO₃ was studiedwith cells of Chlamydomonas reinhardtii which had been grownunder ordinary air (low-CO₂ cells) or air enriched with 4% CO₂(high-CO₂ cells). Experimental data obtained at 20°C andpH 8.0 suggested that the major form of inorganic carbon utilizedby high-CO₂ cells was CO₂, while that utilized by low-CO₂ cellswas HCO₃^–. The cell suspension showed CA activity which was comparableto that observed in the sonicate of cells. Both activities werehigher in low-CO₂ cells than in high-CO₂ cells. The mechanism by which HCO₃^– is utilized by low-CO₂ cellsof C. reinhardtii is discussed. ³Present address: Department of Biology, Faculty of Science,University of Niigata, Niigata 950-21, Japan. (Received August 4, 1982; Accepted January 19, 1983)     

White mutants of Chlamydomonas reinhardtii are defective in phytoene synthase

Carotenoids play an integral and essential role in photosynthesis and photoprotection in plants and algae. A collection of Chlamydomonas reinhardtii mutants lacking carotenoids was characterized for pigment and tocopherol (vitamin E) composition, growth phenotypes under different light conditions, and the molecular basis of their mutant phenotype. The carotenoid-less mutants, or "white" mutants, were also deficient in chlorophylls but had approximately twice the tocopherol content of the wild type. White mutants grew in the dark but were unable to survive in the light, even under very low light conditions on acetate-containing medium. Genetic crosses and recombination tests revealed that all individual white mutants in the collection are alleles of a single gene, lts1, and the white phenotype was closely linked to a marker located in the phytoene synthase gene. DNA sequencing of the phytoene synthase gene from each of the mutants revealed nonsense, missense, frameshift, and splice site mutations. Transformation with a wild-type copy of the phytoene synthase gene was able to complement the lts1-210 mutation. Together, these results show that all the white mutants examined in this work are affected in the phytoene synthase gene.