Biogenic membranes of the chloroplast in Chlamydomonas reinhardtii

The polypeptide subunits of the photosynthetic electron transport complexes in plants and algae are encoded by two genomes. Nuclear genome-encoded subunits are synthesized in the cytoplasm by 80S ribosomes, imported across the chloroplast envelope, and assembled with the subunits that are encoded by the plastid genome. Plastid genome-encoded subunits are synthesized by 70S chloroplast ribosomes directly into membranes that are widely believed to belong to the photosynthetic thylakoid vesicles. However, in situ evidence suggested that subunits of photosystem II are synthesized in specific regions within the chloroplast and cytoplasm of Chlamydomonas. Our results provide biochemical and in situ evidence of biogenic membranes that are localized to these translation zones. A “chloroplast translation membrane” is bound by the translation machinery and appears to be privileged for the synthesis of polypeptides encoded by the plastid genome. Membrane domains of the chloroplast envelope are located adjacent to the cytoplasmic translation zone and enriched in the translocons of the outer and inner chloroplast envelope membranes protein import complexes, suggesting a coordination of protein synthesis and import. Our findings contribute to a current realization that biogenic processes are compartmentalized within organelles and bacteria.     

Inheritance of chloroplast DNA in Chlamydomonas reinhardtii

Two symmetrically located deletions of approximately 100 base pairs each have been identified in chloroplast DNA of Chlamydomonas reinhardtii. Although present in a mutant strain that requires acetate for growth, both deletions have been shown to be distinct from the nonphotosynthetic phenotype of this strain. These physical markers in the chloroplast genome and maternally inherited genetic markers showed strict cotransmission in reciprocal crosses. Thus, our results are consistent with the location of the well-characterized maternally inherited genetic markers in chloroplast DNA of C. reinhardtii.     

Energy-dissipative supercomplex of photosystem II associated with LHCSR3 in Chlamydomonas reinhardtii

Plants and green algae have a low pH-inducible mechanism in photosystem II (PSII) that dissipates excess light energy, measured as the nonphotochemical quenching of chlorophyll fluorescence (qE). Recently, nonphotochemical quenching 4 (npq4), a mutant strain of the green alga Chlamydomonas reinhardtii that is qE-deficient and lacks the light-harvesting complex stress-related protein 3 (LHCSR3), was reported [Peers G, et al. (2009) Nature 462(7272):518–521]. Here, applying a newly established procedure, we isolated the PSII supercomplex and its associated light-harvesting proteins from both WT C. reinhardtii and the npq4 mutant grown in either low light (LL) or high light (HL). LHCSR3 was present in the PSII supercomplex from the HL-grown WT, but not in the supercomplex from the LL-grown WT or mutant. The purified PSII supercomplex containing LHCSR3 exhibited a normal fluorescence lifetime at a neutral pH (7.5) by single-photon counting analysis, but a significantly shorter lifetime at pH 5.5, which mimics the acidified lumen of the thylakoid membranes in HL-exposed chloroplasts. The switch from light-harvesting mode to energy-dissipating mode observed in the LHCSR3-containing PSII supercomplex was sensitive to dicyclohexylcarbodiimide, a protein-modifying agent specific to protonatable amino acid residues. We conclude that the PSII-LHCII-LHCSR3 supercomplex formed in the HL-grown C. reinhardtii cells is capable of energy dissipation on protonation of LHCSR3.     

Perturbation of chloroplast DNA amounts and chloroplast gene transmission in Chlamydomonas reinhardtii by 5-fluorodeoxyuridine

5-Fluorodeoxyuridine selectively decreases the rate of chloroplast DNA replication in Chlamydomonas resulting after several generations of growth in equilibrium levels as low as one-seventh of normal. When the maternal parent is treated prior to mating, the decrease of chloroplast DNA appears to perturb the normal maternal transmission of chloroplast genes, dramatically increasing the proportion of exceptional zygotes transmitting chloroplast genes from the paternal parent.     

State transitions in Chlamydomonas reinhardtii strongly modulate the functional size of photosystem II but not of photosystem I

Plants and green algae optimize photosynthesis in changing light conditions by balancing the amount of light absorbed by photosystems I and II. These photosystems work in series to extract electrons from water and reduce NADP⁺ to NADPH. Light-harvesting complexes (LHCs) are held responsible for maintaining the balance by moving from one photosystem to the other in a process called state transitions. In the green alga Chlamydomonas reinhardtii, a photosynthetic model organism, state transitions are thought to involve 80% of the LHCs. Here, we demonstrate with picosecond-fluorescence spectroscopy on C. reinhardtii cells that, although LHCs indeed detach from photosystem II in state 2 conditions, only a fraction attaches to photosystem I. The detached antenna complexes become protected against photodamage via shortening of the excited-state lifetime. It is discussed how the transition from state 1 to state 2 can protect C. reinhardtii in high-light conditions and how this differs from the situation in plants.Oxygenic photosynthesis is the most important process for fueling life on earth. Light capture and subsequent charge separation processes occur in the so-called photosystems I and II (PSI and PSII). In plants and green algae, both PSs consist of a pigment–protein core complex surrounded by outer light-harvesting complexes (LHCs). Electronic excitations induced by the absorption of sunlight lead to charge separation in the reaction centers (RCs) of PSI and PSII, located in the cores of the PSs. These PSs work in series to extract electrons from water and reduce NADP⁺ to NADPH. For optimal linear electron transport from water to NADP⁺, a balance is needed for the amount of light absorbed by the pigments in the two PSs.Besides carotenoids, the PSII core contains 35 chlorophylls a (Chls a), whereas this number is close to 100 for PSI (1). The outer LHCs consist of various components: The major light-harvesting complex LHCII (a trimer) harbors 12 carotenoids (Cars) and 42 chlorophylls (Chls), 24 of which are Chls a (2), the pigments that are largely responsible for excitation energy transfer (EET) to the PSII RC. In higher plants, there are also three monomeric minor LHCs per core, called CP24, CP26, and CP29, which show high sequence homology with LHCII (see, e.g., ref. 3). In nonstressed conditions, between 85% and 90% of the excitations in PSII lead to charge separation in the RC (4). PSI in plants binds four LHCs (Lhca1–4) (5). The amount of LHCII in the plant membranes is variable and usually ranges from approximately two to approximately four trimers per PSII core, most of which are functionally connected to PSII, although part is also associated with PSI (6). In Chlamydomonas reinhardtii, PSI antenna size differs and there are nine Lhcas per PSI (7). Nine LhcbM genes, plus CP29 and CP26, codify for the antenna complexes of PSII (8), and it was recently shown that, in addition to CP26 and CP29, the PSII supercomplex contains three LHCII trimers per monomeric core (9). In addition, there are usually three to four extra LHCII trimers present per monomeric core (10) (see also below).Although both PSI and PSII contain Chls and Cars, their absorption spectra differ, with PSII being more effective in absorbing blue light and PSI in absorbing far-red light (11–13). Because intensity and spectral composition can vary, organisms need to rapidly adjust the relative absorption cross-sections of both PSs. This regulation occurs via so-called state transitions, and it involves the relocation of Lhcs between PSII and PSI (14).In higher plants, all LHCII is bound to PSII in state 1, whereas in state 2, which can be induced by overexciting PSII, part of LHCII (around 15%) moves to PSI (14, 15). State transitions are regulated by the state of the plastoquinone (PQ) pool via the reversible phosphorylation of LHCII (14, 16–19). The green alga C. reinhardtii, which has widely been used as a model system in photosynthesis research and which might also become important for the production of food and feed ingredients and future biofuels (20), is thought to exhibit state transitions to a far larger extent than higher plants. The widely accepted view is that, during the transition from state 1 to state 2, 80% of the major antenna complexes dissociates from PSII and attaches to PSI (21). This picture is based on results that were obtained with photoacoustic measurements that were used to determine the quantum yield of both PSs in different states. This view has been supported by the finding that the PSII supercomplex is largely disassembled in state 2 (22). However, although a PSI–LHCII supercomplex from C. reinhardtii has been isolated (23), the amount of LHCII associated with it has not been quantified and it has also not been shown that the additional LHCII is capable of transferring energy to the PSI core. More recently, it was argued based on biochemical analysis that also CP26 and CP29 are participating in state transitions in C. reinhardtii (22–25), but again a quantitative analysis is missing.Besides biochemical techniques, time-resolved fluorescence spectroscopy can be helpful to study state transitions and to enlighten the EET processes. The main advantage of this technique is that it can provide both quantitative and functional information for the different states in vivo (6). However, the number of time-resolved fluorescence studies on green algae and especially their state transitions is limited (26–29). Wendler and Holzwarth (27) studied state transitions in the green alga Scenedesmus obliquus using time-resolved fluorescence spectroscopy. They interpreted their data at that time in terms of reversible migration of LHCs between PSII α- and β-centers during state transitions, whereas it was concluded that the size of PSI was not measurably changing (27). Another study was performed by Iwai et al. (28), who used fluorescence lifetime imaging microscopy (FLIM) to visualize state transitions in C. reinhardtii. The authors reported the dissociation of LHCII from PSII during the first part of the transition from state 1 to state 2, but they did not investigate what was happening during the later phase (28). Recently, Wientjes et al. (6) performed a study on light acclimation and state transitions in the plant Arabidopsis thaliana, and among others it was demonstrated quantitatively how time-resolved fluorescence properties of thylakoid membranes change when the relative amount of LHCII attached to PSI and PSII changes (6): When LHCII attaches to PSI, the amplitude of a component with fluorescence lifetime below 100 ps increases significantly, whereas the contribution of the components with lifetimes of several hundreds of picoseconds concomitantly decreases, and the corresponding lifetimes become shorter (6). The former component is mainly due to PSI (with or without LHCII connected) and the latter are due to PSII (with varying amounts of LHCII connected). It is important to point out that the amplitudes of the decay components are directly proportional to the number of pigments that correspond to these decay components (6, 30). Therefore, if during state transitions LHCs are moving from PSII to PSI, then the amplitude(s) of the PSI decay components will increase, whereas those of PSII will decrease. In general, such a reorganization will also lead to some changes in the fluorescence lifetimes. If in addition also quenching processes are introduced, this will lead to an additional change in the lifetime but not in the amplitude (31).Here, we applied time-resolved fluorescence spectroscopy to study changes in PSI and PSII antenna size in response to state transitions for wild-type (WT) C. reinhardtii in vivo. The cells were locked in different states, using the same method as used for previous photoacoustic measurements (21, 32), and fluorescence decay curves were recorded at room temperature. The results lead us to challenge some of the generally accepted views, especially concerning the structure of the PSI supercomplex in state 2 and the fate of the detached LHCII. The main changes during state transitions occur in PSII, whereas changes in the PSI supercomplex turn out to be less pronounced. In addition, it appears that both in state 1 and 2 a pool of LHCII exists that is neither connected to PSI nor to PSII, whereas its size is larger in state 2.     

Engineering the chloroplast genome: techniques and capabilities for chloroplast transformation in Chlamydomonas reinhardtii

Chloroplast transformation of Chlamydomonas reinhardtii has been accomplished by agitating cell wall-deficient cells in the presence of glass beads and DNA. By using the atpB gene as the selected marker and cells grown in 0.5 mM 5-fluorodeoxyuridine, we have recovered up to 50 transformants per microgram of DNA. This method is easy and does not require specialized equipment, although it is not as efficient as the tungsten particle bombardment method [Boynton, J. E., Gillham, N. W., Harris, E. H., Hosler, J. P., Johnson, A. M., Jones, A. R., Randolph-Anderson, B. L., Robertson, D., Klein, T. M., Shark, K. B. & Sanford, J. C. (1988) Science 240, 1534-1537]. By using particle bombardment, we have developed a cotransformation approach in which spectinomycin-resistant 16S rRNA-encoding DNA is the selected marker, and we have demonstrated that cotransformation of an unselected marker on an independent replicon is very efficient. We have used this strategy (i) to recover transformants with partially deleted atpB genes that could not otherwise have been selected since they did not restore photosynthetic capability to a recipient carrying a more extensive atpB deletion and (ii) to generate specific deletion mutations in a wild-type recipient. This methodology should allow the introduction of any desired change into the chloroplast genome, even in the absence of phenotypic selection, and thus a detailed functional analysis of any chloroplast DNA sequence should be possible.     

Structure and function of a chloroplast DNA replication origin of Chlamydomonas reinhardtii

Chloroplast DNA replication in Chlamydomonas reinhardtii is initiated by the formation of a displacement loop (D-loop) at a specific site. One D-loop site with its flanking sequence was cloned in recombinant plasmids SC3-1 and R-13. The sequence of the chloroplast DNA insert in SC3-1, which includes the 0.42-kilobase (kb) D-loop region, as well as 0.2 kb to the 5′ end and 0.43 kb to the 3′ end of the D-loop region, was determined. The sequence is A+T-rich and contains four large stem-loop stuctures. An open reading frame potentially coding for a polypeptide of 136 amino acids was detected in the D-loop region. One stem-loop structure and two back-to-back prokaryotic-type promoters were mapped within the open reading frame. The 5.5-kb EcoRI fragment cloned in R-13 contains the 1.05-kb SC3-1 insert and its flanking regions. A yeast autonomously replicating (ARS) sequence and an ARC sequence, which promotes autonomous replication in Chlamydomonas, have been mapped within the flanking regions [Vallet, J.-M. & Rochaix, J.-D. (1985) Curr. Genet. 9, 321-324]. Both R-13 and SC3-1 were active as templates in a crude algal preparation that supports DNA synthesis. In this in vitro system, chloroplast DNA synthesis initiated near the D-loop site.     

Functional asymmetry of photosystem II D1 and D2 peripheral chlorophyll mutants of Chlamydomonas reinhardtii

The peripheral accessory chlorophylls (Chls) of the photosystem II (PSII) reaction center (RC) are coordinated by a pair of symmetry-related histidine residues (D1-H118 and D2-H117). These Chls participate in energy transfer from the proximal antennae complexes (CP43 and CP47) to the RC core chromophores. In addition, one or both of the peripheral Chls are redox-active and participate in a low-quantum-yield electron transfer cycle around PSII. We demonstrate that conservative mutations of the D2-H117 residue result in decreased Chl fluorescence quenching efficiency attributed to reduced accumulation of the peripheral accessory Chl cation, Chl(Z)(+). In contrast, identical symmetry-related mutations at residue D1-H118 had no effect on Chl fluorescence yield or quenching kinetics. Mutagenesis of the D2-H117 residue also altered the line width of the Chl(Z)(+) EPR signal, but the line shape of the D1-H118Q mutant remained unchanged. The D1-H118 and D2-H117 mutations also altered energy transfer properties in PSII RCs. Unlike wild type or the D1-H118Q mutant, D2-H117N RCs exhibited a reduced CD doublet in the red region of Chl absorbance band, indicative of reduced energetic coupling between P680 and the peripheral accessory Chl. In addition, transient absorption measurements of D2-H117N RCs, excited on the blue side of the Chl absorbance band, exhibited a ( approximately 400 fs) pheophytin Q(X) band bleach lifetime component not seen in wild-type or D1-H118Q RCs. The origin of this component may be related to delayed fast-energy equilibration of the excited state between the core pigments of this mutant.     

Thylakoid membrane polypeptides of Chlamydomonas reinhardtii: wild-type and mutant strains deficient in photosystem II reaction center.

Unstacked thylakoid membrane vesicles were obtained from a homogenate of Chlamydomonas reinhardtii by flotation in a 1.8 M sucrose layer containing 5 mM HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid)-10 mM EDTA (pH 7.5). Sodium dodecyl sulfate-gradient gel electrophoresis showed that the wildtype membranes have a total of at least 33 polypeptides ranging in molecular weights from 68,000 to less than 10,000. The wild-type and three non-photosynthetic mutant strains were studied with respect to their photosynthetic electron transport properties, their fluorescence rise kinetics, and their membrane polypeptide compositions. The results showed a strong correlation between the presence of a membrane polypeptide (molecular weight = 47,000) and the activity of the photosystem II reaction center. This polypeptide is missing from F34 (a mendelian mutant lacking Q, the primary electron acceptor of photosystem II), but is partially restored in a suppressed strain of F34 in which there is an incomplete recovery of photosystem II activity. In a thermosensitive mutant, T4, the same polypeptide is present in reduced amount only in cells grown at 35 degrees but not in those grown at 25 degrees. Evidence from fluorescence rise kinetics and partial photochemical reactions show that the cells grown at 25 degree are similar to wild-type cells but the cells grown at 35 degrees are greatly deficient in Q.     

Extensive sequence homology in the DNA coding for elongation factor Tu from Escherichia coli and the Chlamydomonas reinhardtii chloroplast.

Considerable DNA sequence homology can be detected between the Escherichia coli genes coding for translational components and Chlamydomonas reinhardtii chloroplast DNA. Labeled chloroplast DNA was found to hybridize to restriction fragments of the transducing phage lambda fus3 that code for elongation factor Tu. The chloroplast probe also reacts with fragments coding for ribosomal proteins carried by this phage. The region homologous to the elongation factor genes was located on the physical map of the chloroplast genome by probing restriction fragments of chloroplast DNA with cloned fragments, labeled in vitro, carrying the E. coli elongation factor Tu genes.     

Transmission of chloroplast genes in triploid and tetraploid zygospores of Chlamydomonas reinhardtii: Roles of mating-type gene dosage and gametic chloroplast DNA content

Diploid clones homozygous (mt⁺/mt⁺ or mt^-/mt^-) or heterozygous (mt⁺/mt^-; phenotypically mt^-) for the mating-type locus and homoplasmic for a chloroplast marker conferring resistance to an antibiotic were isolated by artificially induced cell fusion or sexual mating. These diploids were crossed with haploid or diploid strains of opposite mating type and carrying another chloroplast marker. The transmission of the chloroplast genes was analyzed in the triploid and tetraploid zygospores in comparison with diploid zygospores used as controls. The transmission was almost exclusively maternal (mt⁺) (>94%) in the crosses mt⁺ × mt^-, mt⁺/mt⁺ × mt^-, and mt⁺/mt⁺ × mt^-/mt^-. The transmission was preferentially maternal (>76%) in the crosses mt⁺ × mt^-/mt^- whereas in the crosses mt⁺ × mt⁺/mt^-, <50% of the zygospores transmitted the chloroplast allele of maternal (mt⁺) origin. The zygospores produced in crosses mt⁺/mt⁺ × mt⁺/mt^- transmitted the alleles from both parents in >60% of cases. The results show that (i) the presence of one mt⁺ allele in the mt⁺/mt^- (phenotypically mt^-) diploid gametes and (ii) the higher amount of chloroplast DNA molecules (input) present in the diploid gametes versus the haploid ones favor the transmission of the chloroplast allele contributed by these gametes. Moreover, because the zygospores issued from crosses mt⁺/mt⁺ × mt^- and mt⁺ × mt⁺/mt^- were genotypically identical mt⁺/mt⁺/mt^-) but behaved very differently in their chloroplast gene transmission, it was concluded that the molecular events leading to preferential elimination of paternal DNA copies must occur before the fusion of nuclei or chloroplasts in the newly formed zygotes.     

Rapid recovery of chloroplast mutations affecting ribulosebisphosphate carboxylase/oxygenase in Chlamydomonas reinhardtii

Based on the unique ability of chloroplast genes to recombine in Chlamydomonas reinhardtii, a collection of acetate-requiring mutants was screened for recombination with a mutation affecting ribulose-1,5-bisphosphate carboxylase/oxygenase [Rbu-1,5-P₂ carboxylase/oxygenase; 3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39]. This chloroplast mutation, rcl-u-1-10-6C, causes the absence of Rbu-1,5-P₂ carboxylase/oxygenase activities and alters the isoelectric point of the larger subunit. Several mutants that displayed little or no recombination with 10-6C were recovered, and two lacked carboxylase activity. These new chloroplast mutants lack both large and small Rbu-1,5-P₂ carboxylase/oxygenase subunits. The approach demonstrated here permits the routine recovery of chloroplast mutations affecting this enzyme. Multiple mutations in the Rbu-1,5-P₂ carboxylase/oxygenase large-subunit gene can be used to investigate the function and regulation of this enzyme and the regulation of chloroplast genes in general.     

Nucleotide sequence of a multiple-copy gene for the B protein of photosystem II of a cyanobacterium

Chloroplast photogene 32 codes for the 32-kilodalton triazine herbicide-binding protein at the B site of electron transport in photosystem II of the photosynthetic apparatus—its product is the B protein and the gene is accordingly designated ps2B here. The cyanobacteria Anacystis nidulans R2, Fremyella diplosiphon, and Nostoc sp. MAC each contain several copies of ps2B. The sequence of one copy of ps2B from Fremyella, ps2B-1, has been determined. The longest open reading frame would code for a protein of 360 amino acids. Although the deduced amino acid sequence of ps2B-1 is highly homologous overall to that of the corresponding spinach protein [Zurawski, G., Bohnert, H. J., Whitfeld, P. R. & Bottomley, W. (1982) Proc. Natl. Acad. Sci. USA 79, 7699-7703] and, excluding neutral substitutions, the homology is 95% for an internal segment of 309 amino acids, there are a number of nonneutral amino acid substitutions. Most of the differences in net charge and polarity occur in the first 20 amino acids at the amino terminus and in the amino acid composition at the carboxyl terminus. The nucleotide sequences are 76% homologous overall. Conserved sequences resembling prokaryotic “-10” and “-35” regions are found at remarkably similar positions in the spinach and F. diplosiphon sequences although the surrounding sequences show only occasional homologies.     

Identification of the mobile light-harvesting complex II polypeptides for state transitions in Chlamydomonas reinhardtii

State transition in photosynthesis is a short-term balancing mechanism of energy distribution between photosystem I (PSI) and photosystem II (PSII). When PSII is preferentially excited (state 2), a pool of mobile light-harvesting complex II (LHCII) antenna proteins is thought to migrate from PSII to PSI, but biochemical evidence for a physical association between LHCII proteins and PSI in state 2 is weak. Here, using the green alga Chlamydomonas reinhardtii, which has a high capacity for state transitions, we report the isolation of PSI-light-harvesting complex I (LHCI) super-complexes from cells locked into state 1 and state 2. We solubilized the thylakoid membranes with a mild detergent, separated the proteins by sucrose density gradient centrifugation, and subjected gradient fractions to gel-filtration chromatography. Three LHCII polypeptides were associated with a PSI-LHCI supercomplex only in state 2; we identified them as two minor monomeric LHCII proteins (CP26 and CP29) and one previously unreported major LHCII protein type II, or LhcbM5. These three LHCII proteins, in addition to the major trimeric LHCII proteins, were phosphorylated upon transition to state 2. The corresponding phylogenetic tree indicates that among the LHCII proteins associated with PSII, these three LHCII proteins are the most similar to the LHC proteins for PSI (LHCI). Our results are important because CP26, CP29, and LhcbM5, which have been viewed as belonging solely to the PSII complex, are now postulated to shuttle between PSI and PSII during state transitions, thereby acting as docking sites for the trimeric LHCII proteins in both PSI and PSII.