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In chloroplasts, insertion of proteins with multiple transmembrane domains (TMDs) into thylakoid membranes usually occurs in a co-translational manner. Here, we have characterized a thylakoid protein designated FPB1 (Facilitator of PsbB biogenesis1) which together with a previously reported factor PAM68 (Photosynthesis Affected Mutant68) is involved in assisting the biogenesis of CP47, a subunit of the Photosystem II (PSII) core. Analysis by ribosome profiling reveals increased ribosome stalling when the last TMD segment of CP47 emerges from the ribosomal tunnel in fpb1 and pam68. FPB1 interacts with PAM68 and both proteins coimmunoprecipitate with SecY/E and Alb3 as well as with some ribosomal components. Thus, our data indicate that, in coordination with the SecY/E translocon and the Alb3 integrase, FPB1 synergistically cooperates with PAM68 to facilitate the co-translational integration of the last two CP47 TMDs and the large loop between them into thylakoids and the PSII core complex.The assembly of the Photosystem II proximal antenna CP47 remains a challenging question. Here the authors show that FPB1 and PAM68 act coordinately with Alb3 and the SecY/E translocon to facilitate the co-translational integration of specific regions of CP47 into thylakoids. [ABSTRACT FROM AUTHOR]

Chloroplasts rely on the translocon complexes in the outer and inner envelope membranes (the TOC and TIC complexes, respectively) to import thousands of different nuclear-encoded proteins from the cytosol1–4. Although previous studies indicated that the TOC and TIC complexes may assemble into larger supercomplexes5–7, the overall architectures of the TOC–TIC supercomplexes and the mechanism of preprotein translocation are unclear. Here we report the cryo-electron microscopy structure of the TOC–TIC supercomplex from Chlamydomonas reinhardtii. The major subunits of the TOC complex (Toc75, Toc90 and Toc34) and TIC complex (Tic214, Tic20, Tic100 and Tic56), three chloroplast translocon-associated proteins (Ctap3, Ctap4 and Ctap5) and three newly identified small inner-membrane proteins (Simp1–3) have been located in the supercomplex. As the largest protein, Tic214 traverses the inner membrane, the intermembrane space and the outer membrane, connecting the TOC complex with the TIC proteins. An inositol hexaphosphate molecule is located at the Tic214–Toc90 interface and stabilizes their assembly. Four lipid molecules are located within or above an inner-membrane funnel formed by Tic214, Tic20, Simp1 and Ctap5. Multiple potential pathways found in the TOC–TIC supercomplex may support translocation of different substrate preproteins into chloroplasts.A cryo-electron microscopy analysis of the Chlamydomonas reinhardtii TOC–TIC supercomplex reveals that Tic214 traverses the chloroplast inner membrane, the intermembrane space and the outer membrane, connecting the TOC complex with the TIC proteins. [ABSTRACT FROM AUTHOR]

I present my personal reminiscence of Paul Levine—a highly innovative scientist who did seminal work in photosynthesis. He was among the first to initiate and use a genetic approach toward photosynthesis. He greatly helped in establishing the green unicellular alga Chlamydomonas reinhardtii as a powerful model system not only for understanding the function of the photosynthetic apparatus but also for studying its biogenesis and regulation. During the period he spent at Harvard, he made several ground-breaking contributions such as identifying and establishing the order of some components of the photosynthetic electron transport chain as well as determining their genetic origin. He trained many students and post-doctoral fellows several of whom later became prominent in this field and in other areas of plant science. [ABSTRACT FROM AUTHOR]

We present here a Tribute to Klaus Apel (1942-2017), a photosynthesis pioneer—an authority on plant molecular genetics—in five parts. The first section is a prologue. The second section deals with a chronological discussion of Apel’s research life, prepared by the editor Govindjee; it is based on a website article at the Boyce Thompson Institute (BTI) by Patricia Waldron (https://btiscience.org/explore-bti/news/post/bti-says-goodbye-klaus-apel/), as approved for use here by Keith C. Hannon and David Stern of BTI. The third section, which focuses on Apel’s pioneering work on singlet oxygen-mediated EXECUTER-dependent signaling in plants, is written by two of us (J-DR and CK). The fourth section includes three selected reminiscences, one from BTI and two from ETH (Eidgenössische Technische Hochschule). This tribute ends with section five, which is a very brief presentation of Klaus Apel’s personal life, by Wiebke Apel. [ABSTRACT FROM AUTHOR]

In order to maintain optimal photosynthetic activity under a changing light environment, plants and algae need to balance the absorbed light excitation energy between photosystem I and photosystem II through processes called state transitions. Variable light conditions lead to changes in the redox state of the plastoquinone pool which are sensed by a protein kinase closely associated with the cytochrome b f complex. Preferential excitation of photosystem II leads to the activation of the kinase which phosphorylates the light-harvesting system (LHCII), a process which is subsequently followed by the release of LHCII from photosystem II and its migration to photosystem I. The process is reversible as dephosphorylation of LHCII on preferential excitation of photosystem I is followed by the return of LHCII to photosystem II. State transitions involve a considerable remodelling of the thylakoid membranes, and in the case of Chlamydomonas, they allow the cells to switch between linear and cyclic electron flow. In this alga, a major function of state transitions is to adjust the ATP level to cellular demands. Recent studies have identified the thylakoid protein kinase Stt7/STN7 as a key component of the signalling pathways of state transitions and long-term acclimation of the photosynthetic apparatus. In this article, we present a review on recent developments in the area of state transitions. [ABSTRACT FROM AUTHOR]

Photosynthetic organisms are able to adjust to changing light conditions through state transitions, a process that involves the redistribution of light excitation energy between photosystem II (PSII) and photosystem I (PSI). Balancing of the light absorption capacity of these two photosystems is achieved through the reversible association of the major antenna complex (LHCII) between PSII and PSI (ref. 3). Excess stimulation of PSII relative to PSI leads to the reduction of the plastoquinone pool and the activation of a kinase; the phosphorylation of LHCII; and the displacement of LHCII from PSII to PSI (state 2). Oxidation of the plastoquinone pool by excess stimulation of PSI reverses this process (state 1). The Chlamydomonas thylakoid-associated Ser-Thr kinase Stt7, which is required for state transitions, has an orthologue named STN7 in Arabidopsis. Here we show that loss of STN7 blocks state transitions and LHCII phosphorylation. In stn7 mutant plants the plastoquinone pool is more reduced and growth is impaired under changing light conditions, indicating that STN7, and probably state transitions, have an important role in response to environmental changes. [ABSTRACT FROM AUTHOR]

Several membrane proteins were previously shown to bind to the 5′ leader of the chloroplast psbC mRNA in the unicellular eukaryotic alga Chlamydomonas reinhardtii. This study showed that these proteins have affinity for AU-rich RNAs, as determined by competition experiments. In addition, their binding activities are enhanced 13–15-fold by light, and a 46 kDa protein is activated within 1–10 min. This activation could be mediated by the modulation of ADP pools by the light-dependent reactions of photosynthesis and ATP synthase because (1) two inhibitors that block ATP synthesis also prevent this activation and (2) ADP inhibits the RNA-binding activity of this protein in vitro. An inhibitor of Photosystem II diminishes this induction, suggesting that reducing potential generated by the photosynthetic electron transport chain modulates this RNA-binding activity. The RNA-binding activities of two proteins (of 46 and 47 kDa) are inhibited by Mg-protoporphyrin IX methyl ester in vitro suggesting they could be regulated by these intermediates in the chlorophyll biosynthetic pathway. [ABSTRACT FROM AUTHOR]

The sequences of the nuclear genes of the 33 kDa (OEE1) and the 16 kDa (OEE3) polypeptides of the oxygen evolving complex of Chlamydomonas reinhardtii have been established. Comparison between the OEE1 protein sequences of C. reinhardtii and higher plants and cyanobacteria reveals 67 and 47% homology. In contrast, C. reinhardtii and higher plants have only 28% overall homology for OEE3 which is mostly limited to the central portion of the protein. The transit peptides of the C. reinhardtii proteins consist of 52 (OEE1) and, most likely, 51 (OEE1) amino acids. They have a basic amino terminal region and, at least in the case of OEE1, a hydrophobic segment at their carboxy terminal end typical of thylakoid lumen proteins. Comparison of the genomic and cDNA clones indicates that the OEE1 and OEE3 genes contain five and four introns, respectively, some of which are located within the coding sequences of the transit peptides. [ABSTRACT FROM AUTHOR]

cDNA clones encoding two Photosystem I subunits of Chlamydomonas reinhardtii with apparent molecular masses of 18 and 11 kDa (thylakoid polypeptides 21 and 30; P21 and P30 respectively) were isolated using oligonucleotides, the sequences of which were deduced from the N-terminal amino acid sequences of the proteins. The cDNAs were sequenced and used to probe Southern and Northern blots. The Southern blot analysis indicates that both proteins are encoded by single-copy genes. The mRNA sizes of the two components are 1400 and 740 nucleotides, respectively. Comparison between the open reading frames of the cDNAs and the N-terminal amino acid sequences of the proteins indicates that the molecular masses of the mature proteins are 17.9 (P21) and 8.1 kDa (P30). Analysis of the deduced protein sequences predicts that both subunits are extrinsic membrane proteins with net positive charges. The amino acid sequences of the transit peptides suggest that P21 and P30 are routed towards the lumenal and stromal sides of the thylakoid membranes, respectively. [ABSTRACT FROM AUTHOR]

Using particle gun-mediated chloroplast transformation we have disrupted the psbK gene of Chlamydomonas reihardtii with an aadA expression cassette that confers resistance to spectinomycin. The transformants are unable to grow photoautotrophically, but they grow normally in acetate-containing medium. They are deficient in photosystem II activity as measured by fluorescence transients and O evolution and they accumulate less than 10% of wild-type levels of photosystem II as measured by immunochemical means. Pulse-labeling experiments indicate that the photosystem II complex is synthesized normally in the transformants. These results differ from those obtained previously with similar cyanobacterial psbK mutants that were still capable of photoautotrophic growth (Ikeuchi et al., J. Biol. Chem. 266 (1991) 1111-1115). In C. reinhardtii the psbK product is required for the stable assembly and/or stability of the photosystem II complex and essential for photoautotrophic growth. The data also suggest that the stability requirements of the photosynthetic complexes differ considerably between C. reinhardtii and cyanobacteria. [ABSTRACT FROM AUTHOR]

We report the rescue of an arginine-requiring mutant ( arg7-8) of Chlamydomonas reinhardtii by complementation using total DNA from a genomic cosmid library. Using the glass-bead transformation method of Kindle [8] four putative transformants able to grow in the absence of exogenous arginine were obtained from 3×10 treated cells. Southern blot analysis reveals that at least three of the clones have acquired an additional copy of the gene ( ARG7) encoding argininosuccinate lyase (ASL). The arginine-independent phenotype is stable in the absence of selective pressure and high levels of ASL activity are detected in all four clones. We conclude that these represent true transformants and that any stable nuclear mutant of Chlamydomonas could be rescued using this approach. [ABSTRACT FROM AUTHOR]

In the secondary structure model that has been proposed for the trans-spliced intron 1 in the Chlamydomonas reinhardtii psaA gene, a third RNA species ( tscA RNA) interacts with the 5′ and 3′ intron parts flanking the exons to reconstitute a composite structure with several features of group-II introns. To test the validity of this model, we undertook the sequencing and modelling of equivalent introns in the psaA gene from other unicellular green algae belonging to the highly diversified genus Chlamydomonas. Our comparative analysis supports the model reported for the C. reinhardtii psaA intron 1, and also indicates that the 5′ end of the tscA RNA and the region downstream from the psaA exon 1 cannot be folded into a structure typical of domain I as described for most group-II introns. It is possible that a fourth RNA species, yet to be discovered, provides the parts of domain I which are apparently missing. [ABSTRACT FROM AUTHOR]

The introduction of exogenous DNA into the nuclear genome of Chlamydomonas reinhardtii occurs predominantly via non-homologous (illegitimate) recombination and results in integration at apparently-random loci. Using truncated and modified versions of the C. reinhardtii ARG7 gene in a series of transformation experiments, we demonstrate that homologous recombination between introduced DNA molecules occurs readily in C. reinhardtii, requires a region of homology of no more than 230 bp, and gives rise to intact copies of ARG7 in the nuclear genome. Evidence is presented for homologous recombination between introduced ARG7 DNA and the resident copy of the gene, and for the de-novo synthesis of the ARG7 sequence during transformation. [ABSTRACT FROM AUTHOR]

Four cDNAs (cDNAs 1-4),162,338,321 and 167 by in size, that contain repetitive DNA sequences, were isolated from C. reinhardtii. cDNAs 1, 2 and 3 hybridized to multiple transcripts in poly A RNA. Each of the four repeat families is comprised of an extremely heterogeneous population of interspersed nuclear DNA sequences most of which are less than 0.5 kbp in size. A large number of restriction fragment length polymorphisms were uncovered by using cDNAs 1 and 2 as hybridization probes. cDNA2 was compared to two different genomic DNA sequences: the first sequence was complementary to a central 136 bases of cDNA2, which is bordered by a 15-bp imperfect direct repeat; the second sequence lacks a poly-dA tail, but is otherwise colinear along its entire length with cDNA2. This suggests that some members of the cDNA2 repeat family contain signals for polyadenylation. The majority of accumulated transcripts that hybridize to cDNA2 have the same 5′-3′ orientation as cDNA2. [ABSTRACT FROM AUTHOR]

A 5.3 kb chloroplast restriction fragment of Chlamydomonas reinhardii containing an origin of DNA replication and a sequence capable of promoting autonomous replication in C. reinhardii (ARC sequence) also carries an ARS sequence (autonomous replication in yeast). The ARC and ARS elements have been physically mapped and shown to be distinct from the origin of DNA replication. Similarly, restriction fragments containing the origin of chloroplast DNA replication from Euglena gracilis are unable to promote autonomous replication in yeast. [ABSTRACT FROM AUTHOR]

The organization of the nuclear ribosomal units of five different species of Chlamydomonas has been examined by hybridization of their nuclear DNA fragments produced by several restriction endonucleases with a radioactively labelled probe consisting of the two cloned BamHI ribosomal fragments of C. reinhardii. The results indicate that a) the ribosomal units of these five species are structurally related, b) changes in the non transcribed spacer occur in C. eugametos and in C. globosa, c) the rDNA unit of C. intermedia contains either an enlarged internal transcribed spacer or a ribosomal intervening sequence, d) the rDNA units of C. reinhardii and C. callosa are indistinguishable. [ABSTRACT FROM AUTHOR]

Pulse labelled rDNA from cultured somatic cells of Xenopus laevis was examined by electron microscope autoradiography. The pattern of replication closely resembles that of bulk chromosomal DNA and differs considerably from rDNA synthesis during amplification in the oocyte. - About 0.15% of the rDNA molecules in the purified preparations were circular. The presence of interlocked circles of equal size indicates that the circles are not in vitro cyclization artefacts, but may represent free rRNA genes. A low frequency of circles was also seen in Xenopus blood rDNA. Their stability in high concentration of formamide suggests that they too did not arise after DNA extraction. [ABSTRACT FROM AUTHOR]

We have isolated complementary DNA (cDNA) clones for apocytochrome c from the green alga Chlamydomonas reinhardtii and shown that they are encoded by a single nuclear gene termed cyc. Cyc mRNA levels are found to depend primarily on the presence of acetate as a reduced carbon source in the culture medium. The deduced amino acid sequence shows that, apart from the probable removal of the initiating methionine, C. reinhardtii apocytochrome c is syntheszed in its mature form. Its structure is generally similar to that of cytochromes c from higher plants. Several punctual deviations from the general pattern of cytochrome c sequences that is found in other organisms have interesting structural and functional implications. These include, in particular, valines 19 and 39, asparagine 78, and alanine 83. A phylogenetic tree was constructed by the matrix method from cytochrome c data for a representative range of species. The results suggest that C. reinhardtii diverged from higher plants approximately 700-750 million years ago; they also are not easy to reconcile with the current attribution of Chlamydomonas reinhardtii and Enteromorpha intestinalis to a unique phylum, because these two species probably diverged from one another at about the same time as they diverged from the line leading to higher plants. [ABSTRACT FROM AUTHOR]

The ribulosebisphosphate carboxylase/oxygenase-defective Chlamydomonas mutant, 10-6C, was the first mutant to be physically defined in chloroplast DNA. In this report, a photosynthesis-competent revertant of the 10-6C mutant has been found to result from true reversion within the chloroplast large-subunit gene. This result supports the original assignment of the 10-6C mutation within the large-subunit gene. [ABSTRACT FROM AUTHOR]