Chloroplast biogenesis involves spatial coordination of nuclear and organellar gene expression in Chlamydomonas.

The localization of translation can direct the polypeptide product to the proper intracellular compartment. Our results reveal translation by cytosolic ribosomes on a domain of the chloroplast envelope in the unicellular green alga Chlamydomonas (Chlamydomonas reinhardtii). We show that this envelope domain of isolated chloroplasts retains translationally active ribosomes and mRNAs encoding chloroplast proteins. This domain is aligned with localized translation by chloroplast ribosomes in the translation zone, a chloroplast compartment where photosystem subunits encoded by the plastid genome are synthesized and assembled. Roles of localized translation in directing newly synthesized subunits of photosynthesis complexes to discrete regions within the chloroplast for their assembly are suggested by differences in localization on the chloroplast of mRNAs encoding either subunit of the light-harvesting complex II or the small subunit of Rubisco. Transcription of the chloroplast genome is spatially coordinated with translation, as revealed by our demonstration of a subpopulation of transcriptionally active chloroplast nucleoids at the translation zone. We propose that the expression of chloroplast proteins by the nuclear-cytosolic and organellar genetic systems is organized in spatially aligned subcompartments of the cytoplasm and chloroplast to facilitate the biogenesis of the photosynthetic complexes.

Plastid Nucleoids: Insights into Their Shape and Dynamics.

Chloroplasts/Plastids are unique organelles found in plant cells and some algae, and are responsible for performing essential functions such as photosynthesis. The plastid genome, consisting of circular and linear DNA molecules, is packaged and organized into specialized structures called nucleoids. The composition and dynamics of these nucleoids have been the subject of intense research, as they are critical for proper plastid functions and development. In this minireview, recent advances in understanding the organization and regulation of plastid nucleoids are overviewed, with a focus on the various proteins and factors that regulate the shape and dynamics of nucleoids, including DNA-binding proteins, and membrane anchorage proteins. The dynamic nature of nucleoid organization, which is influenced by a variety of developmental cues and the cell cycle, is also examined.

A conserved RWP-RK transcription factor VSR1 controls gametic differentiation in volvocine algae.

Volvocine green algae are a model for understanding the evolution of mating types and sexes. They are facultatively sexual, with gametic differentiation occurring in response to nitrogen starvation (-N) in most genera and to sex inducer hormone in Volvox. The conserved RWP-RK family transcription factor (TF) MID is encoded by the minus mating-type locus or male sex-determining region of heterothallic volvocine species and dominantly determines minus or male gametic differentiation. However, the factor(s) responsible for establishing the default plus or female differentiation programs have remained elusive. We performed a phylo-transcriptomic screen for autosomal RWP-RK TFs induced during gametogenesis in unicellular isogamous Chlamydomonas reinhardtii (Chlamydomonas) and in multicellular oogamous Volvox carteri (Volvox) and identified a single conserved ortho-group we named Volvocine Sex Regulator 1 (VSR1). Chlamydomonas vsr1 mutants of either mating type failed to mate and could not induce expression of key mating-type-specific genes. Similarly, Volvox vsr1 mutants in either sex could initiate sexual embryogenesis, but the presumptive eggs or androgonidia (sperm packet precursors) were infertile and unable to express key sex-specific genes. Yeast two-hybrid assays identified a conserved domain in VSR1 capable of self-interaction or interaction with the conserved N terminal domain of MID. In vivo coimmunoprecipitation experiments demonstrated association of VSR1 and MID in both Chlamydomonas and Volvox. These data support a new model for volvocine sexual differentiation where VSR1 homodimers activate expression of plus/female gamete-specific-genes, but when MID is present, MID-VSR1 heterodimers are preferentially formed and activate minus/male gamete-specific-genes.

HBD1 protein with a tandem repeat of two HMG-box domains is a DNA clip to organize chloroplast nucleoids in Chlamydomonas reinhardtii.

Compaction of bulky DNA is a universal issue for all DNA-based life forms. Chloroplast nucleoids (chloroplast DNA-protein complexes) are critical for chloroplast DNA maintenance and transcription, thereby supporting photosynthesis, but their detailed structure remains enigmatic. Our proteomic analysis of chloroplast nucleoids of the green alga Chlamydomonas reinhardtii identified a protein (HBD1) with a tandem repeat of two DNA-binding high mobility group box (HMG-box) domains, which is structurally similar to major mitochondrial nucleoid proteins transcription factor A, mitochondrial (TFAM), and ARS binding factor 2 protein (Abf2p). Disruption of the HBD1 gene by CRISPR-Cas9-mediated genome editing resulted in the scattering of chloroplast nucleoids. This phenotype was complemented when intact HBD1 was reintroduced, whereas a truncated HBD1 with a single HMG-box domain failed to complement the phenotype. Furthermore, ectopic expression of HBD1 in the mitochondria of yeast Δabf2 mutant successfully complemented the defects, suggesting functional similarity between HBD1 and Abf2p. Furthermore, in vitro assays of HBD1, including the electrophoretic mobility shift assay and DNA origami/atomic force microscopy, showed that HBD1 is capable of introducing U-turns and cross-strand bridges, indicating that proteins with two HMG-box domains would function as DNA clips to compact DNA in both chloroplast and mitochondrial nucleoids.

Three genomes in the algal genus Volvox reveal the fate of a haploid sex-determining region after a transition to homothallism.

Transitions between separate sexes (dioecy) and other mating systems are common across eukaryotes. Here, we study a change in a haploid dioecious green algal species with male- and female-determining chromosomes (U and V). The genus Volvox is an oogamous (with large, immotile female gametes and small, motile male gametes) and includes both heterothallic species (with distinct male and female genotypes, associated with a mating-type system that prevents fusion of gametes of the same sex) and homothallic species (bisexual, with the ability to self-fertilize). We date the origin of an expanded sex-determining region (SDR) in Volvox to at least 75 Mya, suggesting that homothallism represents a breakdown of dioecy (heterothallism). We investigated the involvement of the SDR of the U and V chromosomes in this transition. Using de novo whole-genome sequences, we identified a heteromorphic SDR of ca 1 Mbp in male and female genotypes of the heterothallic species Volvox reticuliferus and a homologous region (SDLR) in the closely related homothallic species Volvox africanus, which retained several different hallmark features of an SDR. The V. africanus SDLR includes a large region resembling the female SDR of the presumptive heterothallic ancestor, whereas most genes from the male SDR are absent. However, we found a multicopy array of the male-determining gene, MID, in a different genomic location from the SDLR. Thus, in V. africanus, an ancestrally female genotype may have acquired MID and thereby gained male traits.

Chloroplast proteotoxic stress-induced autophagy is involved in the degradation of chloroplast proteins in Chlamydomonas reinhardtii.

Intraorganellar proteases and cytoplasmic proteolytic systems such as autophagy orchestrate the degradation of organellar proteins to ensure organelle homeostasis in eukaryotic cells. The green alga Chlamydomonas reinhardtii is an ideal unicellular model organism for elucidating the mechanisms maintaining proteostasis in chloroplasts. However, the autophagic pathways targeting the photosynthetic organelles of these algae have not been clearly elucidated. Here, we explored the role of autophagy in chloroplast protein degradation in Chlamydomonas cells. We labeled the chloroplast protein Rubisco small subunit (RBCS) with the yellow fluorescent protein Venus in a Chlamydomonas strain in which expression of the chloroplast gene clpP1, encoding a major catalytic subunit of the chloroplast Clp protease, can be conditionally repressed to selectively perturb chloroplast protein homeostasis. We observed transport of both nucleus-encoded RBCS-Venus fusion protein and chloroplast-encoded Rubisco large subunit (rbcL) from the chloroplast to the vacuoles in response to chloroplast proteotoxic stress induced by clpP1 inhibition. This process was retarded by the addition of autophagy inhibitors. Biochemical detection of lytic cleavage of RBCS-Venus supported the notion that Rubisco is degraded in the vacuoles via autophagy. Electron microscopy revealed vacuolar accumulation of autophagic vesicles and exposed their ultrastructure during repression of clpP1 expression. Treatment with an autophagy activator also induced chloroplast autophagy. These results indicate that autophagy contributes to chloroplast protein degradation in Chlamydomonas cells.

The Pentatricopeptide Repeat Protein PGR3 Is Required for the Translation of petL and ndhG by Binding Their 5' UTRs.

PGR3 is a P-class pentatricopeptide repeat (PPR) protein required for the stabilization of petL operon RNA and the translation of the petL gene in plastids. Irrespective of its important roles in plastids, key questions have remained unanswered, including how PGR3 protein promotes translation and which plastid mRNA PGR3 activates the translation. Here, we show that PGR3 facilitates the translation from ndhG, in addition to petL, through binding to their 5' untranslated regions (UTRs). Ribosome profiling and RNA sequencing in pgr3 mutants revealed that translation from petL and ndhG was specifically suppressed. Harnessing small RNA fragments protected by PPR proteins in vivo, we probed the PGR3 recruitment to the 5' UTRs of petL and ndhG. The putative PGR3-bound RNA segments per se repress the translation possibly with a strong secondary structure and thereby block ribosomes' access. However, the PGR3 binding antagonizes the effects and facilitates the protein synthesis from petL and ndhG in vitro. The prediction of the 3-dimensional structure of PGR3 suggests that the 26th PPR motif plays important roles in target RNA binding. Our data show the specificity of a plastidic RNA-binding protein and provide a mechanistic insight into translational control.

Peroxisome Deficiency Impairs BDNF Signaling and Memory.

Peroxisome is an intracellular organelle that functions in essential metabolic pathways including ß-oxidation of very-long-chain fatty acids and biosynthesis of plasmalogens. Peroxisome biogenesis disorders (PBDs) manifest severe dysfunction in multiple organs including central nervous system (CNS), whilst the pathogenic mechanisms are largely unknown. We recently reported that peroxisome-deficient neural cells secrete an increased level of brain-derived neurotrophic factor (BDNF), resulting in the cerebellar malformation. Peroxisomal functions in adulthood brain have been little investigated. To induce the peroxisome deficiency in adulthood brain, we here established tamoxifen-inducible conditional Pex2-knockout mouse. Peroxisome deficiency in the conditional Pex2-knockout adult mouse brain induces the upregulated expression of BDNF and its inactive receptor TrkB-T1 in hippocampus, which notably results in memory disturbance. Our results suggest that peroxisome deficiency gives rise to the dysfunction of hippocampal circuit via the impaired BDNF signaling.

Holliday Junction Resolvase MOC1 Maintains Plastid and Mitochondrial Genome Integrity in Algae and Bryophytes.

When DNA double-strand breaks occur, four-stranded DNA structures called Holliday junctions (HJs) form during homologous recombination. Because HJs connect homologous DNA by a covalent link, resolution of HJ is crucial to terminate homologous recombination and segregate the pair of DNA molecules faithfully. We recently identified Monokaryotic Chloroplast1 (MOC1) as a plastid DNA HJ resolvase in algae and plants. Although Cruciform cutting endonuclease1 (CCE1) was identified as a mitochondrial DNA HJ resolvase in yeasts, homologs or other mitochondrial HJ resolvases have not been identified in other eukaryotes. Here, we demonstrate that MOC1 depletion in the green alga Chlamydomonas reinhardtii and the moss Physcomitrella patens induced ectopic recombination between short dispersed repeats in ptDNA. In addition, MOC1 depletion disorganized thylakoid membranes in plastids. In some land plant lineages, such as the moss P. patens, a liverwort and a fern, MOC1 dually targeted to plastids and mitochondria. Moreover, mitochondrial targeting of MOC1 was also predicted in charophyte algae and some land plant species. Besides causing instability of plastid DNA, MOC1 depletion in P. patens induced short dispersed repeat-mediated ectopic recombination in mitochondrial DNA and disorganized cristae in mitochondria. Similar phenotypes in plastids and mitochondria were previously observed in mutants of plastid-targeted (RECA2) and mitochondrion-targeted (RECA1) recombinases, respectively. These results suggest that MOC1 functions in the double-strand break repair in which a recombinase generates HJs and MOC1 resolves HJs in mitochondria of some lineages of algae and plants as well as in plastids in algae and plants.

A non-photosynthetic green alga illuminates the reductive evolution of plastid electron transport systems.

BACKGROUND: Plastid electron transport systems are essential not only for photosynthesis but also for dissipating excess reducing power and sinking excess electrons generated by various redox reactions. Although numerous organisms with plastids have lost their photoautotrophic lifestyles, there is a spectrum of known functions of remnant plastids in non-photosynthetic algal/plant lineages; some of non-photosynthetic plastids still retain diverse metabolic pathways involving redox reactions while others, such as apicoplasts of apicomplexan parasites, possess highly reduced sets of functions. However, little is known about underlying mechanisms for redox homeostasis in functionally versatile non-photosynthetic plastids and thus about the reductive evolution of plastid electron transport systems. RESULTS: Here we demonstrated that the central component for plastid electron transport systems, plastoquinone/plastoquinol pool, is still retained in a novel strain of an obligate heterotrophic green alga lacking the photosynthesis-related thylakoid membrane complexes. Microscopic and genome analyses revealed that the Volvocales green alga, chlamydomonad sp. strain NrCl902, has non-photosynthetic plastids and a plastid DNA that carries no genes for the photosynthetic electron transport system. Transcriptome-based in silico prediction of the metabolic map followed by liquid chromatography analyses demonstrated carotenoid and plastoquinol synthesis, but no trace of chlorophyll pigments in the non-photosynthetic green alga. Transient RNA interference knockdown leads to suppression of plastoquinone/plastoquinol synthesis. The alga appears to possess genes for an electron sink system mediated by plastid terminal oxidase, plastoquinone/plastoquinol, and type II NADH dehydrogenase. Other non-photosynthetic algae/land plants also possess key genes for this system, suggesting a broad distribution of an electron sink system in non-photosynthetic plastids. CONCLUSION: The plastoquinone/plastoquinol pool and thus the involved electron transport systems reported herein might be retained for redox homeostasis and might represent an intermediate step towards a more reduced set of the electron transport system in many non-photosynthetic plastids. Our findings illuminate a broadly distributed but previously hidden step of reductive evolution of plastid electron transport systems after the loss of photosynthesis.

Step-wise elimination of α-mitochondrial nucleoids and mitochondrial structure as a basis for the strict uniparental inheritance in Cryptococcus neoformans.

In most sexual eukaryotes, mitochondrial (mt) DNA is uniparentally inherited, although the detailed mechanisms underlying this phenomenon remain controversial. The most widely accepted explanations include the autophagic elimination of paternal mitochondria in the fertilized eggs and the active degradation of paternal mitochondrial DNA. To decode the precise program for the uniparental inheritance, we focused on Cryptococcus neoformans as a model system, in which mtDNA is inherited only from the a-parent, although gametes of a- and α-cells are of equal size and contribute equal amounts of mtDNA to the zygote. In this research, the process of preferential elimination of the mitochondria contributed by the α-parent (α-mitochondria) was studied by fluorescence microscopy and single cell analysis using optical tweezers, which revealed that α-mitochondria are preferentially reduced by the following three steps: (1) preferential reduction of α-mitochondrial (mt) nucleoids and α-mtDNA, (2) degradation of the α-mitochondrial structure and (3) proliferation of remaining mt nucleoids during the zygote development. Furthermore, AUTOPHAGY RELATED GENE (ATG) 8 and the gene encoding mitochondrial endonuclease G (NUC1) were disrupted, and the effects of their disruption on the uniparental inheritance were scrutinized. Disruption of ATG8 (ATG7) and NUC1 did not have severe effects on the uniparental inheritance, but microscopic examination revealed that α-mitochondria lacking mt nucleoids persisted in Δatg8 zygotes, indicating that autophagy is not critical for the uniparental inheritance per se but is responsible for the clearance of mitochondrial structures after the reduction of α-mt nucleoids.

Isolation and Characterization of Chlamydomonas Autophagy-Related Mutants in Nutrient-Deficient Conditions.

Autophagy is a recycling system for amino acids and carbon- and nitrogen (N)-containing compounds. To date, the functional importance of autophagy in microalgae in nutrient-deficient conditions has not been evaluated by using autophagy-defective mutants. Here, we provide evidence which supports the following notions by characterizing an insertional mutant of the autophagy-related gene ATG8, encoding a ubiquitin-like protein necessary for the formation of the autophagosome in the green alga, Chlamydomonas reinhardtii. First, ATG8 is required for maintenance of cell survival and Chl content in N-, sulfur- and phosphate-deficient conditions. Secondly, ATG8 supports the degradation of triacylglycerol and lipid droplets after the resupply of N to cells cultured in N-limiting conditions. Thirdly, ATG8 is also necessary for accumulation of starch in phosphate-deficient conditions. Additionally, autophagy is not essential for maternal inheritance of the organelle genomes in gametogenesis.

Chloroplast nucleoids as a transformable network revealed by live imaging with a microfluidic device.

Chloroplast DNA is organized into DNA-protein conglomerates called chloroplast nucleoids, which are replicated, transcribed, and inherited. We applied live-imaging technology with a microfluidic device to examine the nature of chloroplast nucleoids in Chlamydomonas reinhardtii. We observed the dynamic and reversible dispersion of globular chloroplast nucleoids into a network structure in dividing chloroplasts. In the monokaryotic chloroplast (moc) mutant, in which chloroplast nucleoids are unequally distributed following chloroplast division due to a defect in MOC1, the early stages of chloroplast nucleoid formation occurred mainly in the proximal area. This suggests the chloroplast nucleoid transformable network consists of a highly compact core with proximal areas associated with cpDNA replication and nucleoid formation.

Gene Regulatory Networks for the Haploid-to-Diploid Transition of Chlamydomonas reinhardtii.

The sexual cycle of the unicellular Chlamydomonas reinhardtii culminates in the formation of diploid zygotes that differentiate into dormant spores that eventually undergo meiosis. Mating between gametes induces rapid cell wall shedding via the enzyme g-lysin; cell fusion is followed by heterodimerization of sex-specific homeobox transcription factors, GSM1 and GSP1, and initiation of zygote-specific gene expression. To investigate the genetic underpinnings of the zygote developmental pathway, we performed comparative transcriptome analysis of both pre- and post-fertilization samples. We identified 253 transcripts specifically enriched in early zygotes, 82% of which were not up-regulated in gsp1 null zygotes. We also found that the GSM1/GSP1 heterodimer negatively regulates the vegetative wall program at the posttranscriptional level, enabling prompt transition from vegetative wall to zygotic wall assembly. Annotation of the g-lysin-induced and early zygote genes reveals distinct vegetative and zygotic wall programs, supported by concerted up-regulation of genes encoding cell wall-modifying enzymes and proteins involved in nucleotide-sugar metabolism. The haploid-to-diploid transition in Chlamydomonas is masterfully controlled by the GSM1/GSP1 heterodimer, translating fertilization and gamete coalescence into a bona fide differentiation program. The fertilization-triggered integration of genes required to make related, but structurally and functionally distinct organelles-the vegetative versus zygote cell wall-presents a likely scenario for the evolution of complex developmental gene regulatory networks.

Holliday junction resolvases mediate chloroplast nucleoid segregation.

Holliday junctions, four-stranded DNA structures formed during homologous recombination, are disentangled by resolvases that have been found in prokaryotes and eukaryotes but not in plant organelles. Here, we identify monokaryotic chloroplast 1 (MOC1) as a Holliday junction resolvase in chloroplasts by analyzing a green alga Chlamydomonas reinhardtii mutant defective in chloroplast nucleoid (DNA-protein complex) segregation. MOC1 is structurally similar to a bacterial Holliday junction resolvase, resistance to ultraviolet (Ruv) C, and genetically conserved among green plants. Reduced or no expression of MOC1 in Arabidopsis thaliana leads to growth defects and aberrant chloroplast nucleoid segregation. In vitro biochemical analysis and high-speed atomic force microscopic analysis revealed that A. thaliana MOC 1 (AtMOC1) binds and cleaves the core of Holliday junctions symmetrically. MOC1 may mediate chloroplast nucleoid segregation in green plants by resolving Holliday junctions.

Dynamic Interplay between Nucleoid Segregation and Genome Integrity in Chlamydomonas Chloroplasts.

The chloroplast (cp) genome is organized as nucleoids that are dispersed throughout the cp stroma. Previously, a cp homolog of bacterial recombinase RecA (cpRECA) was shown to be involved in the maintenance of cp genome integrity by repairing damaged chloroplast DNA and by suppressing aberrant recombination between short dispersed repeats in the moss Physcomitrella patens Here, overexpression and knockdown analysis of cpRECA in the green alga Chlamydomonas reinhardtii revealed that cpRECA was involved in cp nucleoid dynamics as well as having a role in maintaining cp genome integrity. Overexpression of cpRECA tagged with yellow fluorescent protein or hemagglutinin resulted in the formation of giant filamentous structures that colocalized exclusively to chloroplast DNA and cpRECA localized to cp nucleoids in a heterogenous manner. Knockdown of cpRECA led to a significant reduction in cp nucleoid number that was accompanied by nucleoid enlargement. This phenotype resembled those of gyrase inhibitor-treated cells and monokaryotic chloroplast mutant cells and suggested that cpRECA was involved in organizing cp nucleoid dynamics. The cp genome also was destabilized by induced recombination between short dispersed repeats in cpRECA-knockdown cells and gyrase inhibitor-treated cells. Taken together, these results suggest that cpRECA and gyrase are both involved in nucleoid dynamics and the maintenance of genome integrity and that the mechanisms underlying these processes may be intimately related in C. reinhardtii cps.

C-Terminal Region of Sulfite Reductase Is Important to Localize to Chloroplast Nucleoids in Land Plants.

Chloroplast (cp) DNA is compacted into cpDNA-protein complexes, called cp nucleoids. An abundant and extensively studied component of cp nucleoids is the bifunctional protein sulfite reductase (SiR). The preconceived role of SiR as the core cp nucleoid protein, however, is becoming less likely because of the recent findings that SiRs do not associate with cp nucleoids in some plant species, such as Zea mays and Arabidopsis thaliana To address this discrepancy, we have performed a detailed phylogenetic analysis of SiRs, which shows that cp nucleoid-type SiRs share conserved C-terminally encoded peptides (CEPs). The CEPs are likely to form a bacterial ribbon-helix-helix DNA-binding motif, implying a potential role in attaching SiRs onto cp nucleoids. A proof-of-concept experiment was conducted by fusing the nonnucleoid-type SiR from A. thaliana (AtSiR) with the CEP from the cp nucleoid-type SiR of Phaseolus vulgaris The addition of the CEP drastically altered the intra-cp localization of AtSiR to cp nucleoids. Our analysis supports the possible functions of CEPs in determining the localization of SiRs to cp nucleoids and illuminates a possible evolutionary scenario for SiR as a cp nucleoid protein.

Sequence of the Gonium pectorale Mating Locus Reveals a Complex and Dynamic History of Changes in Volvocine Algal Mating Haplotypes.

Sex-determining regions (SDRs) or mating-type (MT) loci in two sequenced volvocine algal species, Chlamydomonas reinhardtii and Volvox carteri, exhibit major differences in size, structure, gene content, and gametolog differentiation. Understanding the origin of these differences requires investigation of MT loci from related species. Here, we determined the sequences of the minus and plus MT haplotypes of the isogamous 16-celled volvocine alga, Gonium pectorale, which is more closely related to the multicellular V. carteri than to C. reinhardtii Compared to C. reinhardtii MT, G. pectorale MT is moderately larger in size, and has a less complex structure, with only two major syntenic blocs of collinear gametologs. However, the gametolog content of G. pectorale MT has more overlap with that of V. carteri MT than with C. reinhardtii MT, while the allelic divergence between gametologs in G. pectorale is even lower than that in C. reinhardtii Three key sex-related genes are conserved in G. pectorale MT: GpMID and GpMTD1 in MT-, and GpFUS1 in MT+. GpFUS1 protein exhibited specific localization at the plus-gametic mating structure, indicating a conserved function in fertilization. Our results suggest that the G. pectorale-V. carteri common ancestral MT experienced at least one major reformation after the split from C. reinhardtii, and that the V. carteri ancestral MT underwent a subsequent expansion and loss of recombination after the divergence from G. pectorale These data begin to polarize important changes that occurred in volvocine MT loci, and highlight the potential for discontinuous and dynamic evolution in SDRs.

A Cyan Fluorescent Reporter Expressed from the Chloroplast Genome of Marchantia polymorpha.

Recently, the liverwort Marchantia polymorpha has received increasing attention as a basal plant model for multicellular studies. Its ease of handling, well-characterized plastome and proven protocols for biolistic plastid transformation qualify M. polymorpha as an attractive platform to study the evolution of chloroplasts during the transition from water to land. In addition, chloroplasts of M. polymorpha provide a convenient test-bed for the characterization of genetic elements involved in plastid gene expression due to the absence of mechanisms for RNA editing. While reporter genes have proven valuable to the qualitative and quantitative study of gene expression in chloroplasts, expression of green fluorescent protein (GFP) in chloroplasts of M. polymorpha has proven problematic. We report the design of a codon-optimized gfp varian, mturq2cp, which allowed successful expression of a cyan fluorescent protein under control of the tobacco psbA promoter from the chloroplast genome of M. polymorpha. We demonstrate the utility of mturq2cp in (i) early screening for transplastomic events following biolistic transformation of M. polymorpha spores; (ii) visualization of stromules as elements of plastid structure in Marchantia; and (iii) quantitative microscopy for the analysis of promoter activity.

Eukaryotic Components Remodeled Chloroplast Nucleoid Organization during the Green Plant Evolution.

Chloroplast (cp) DNA is thought to originate from the ancestral endosymbiont genome and is compacted to form nucleoprotein complexes, cp nucleoids. The structure of cp nucleoids is ubiquitously observed in diverse plants from unicellular algae to flowering plants and is believed to be a multifunctional platform for various processes, including cpDNA replication, repair/recombination, transcription, and inheritance. Despite its fundamental functions, the protein composition for cp nucleoids in flowering plants was suggested to be divergent from those of bacteria and algae, but the evolutionary process remains elusive. In this research, we aimed to reveal the evolutionary history of cp nucleoid organization by analyzing the key organisms representing the three evolutionary stages of eukaryotic phototrophs: the chlorophyte alga Chlamydomonas reinhardtii, the charophyte alga Klebsormidium flaccidum, and the most basal land plant Marchantia polymorpha. To clarify the core cp nucleoid proteins in C. reinhardtii, we performed an LC-MS/MS analysis using highly purified cp nucleoid fractions and identified a novel SAP domain-containing protein with a eukaryotic origin as a constitutive core component. Then, homologous genes for cp nucleoid proteins were searched for in C. reinhardtii, K. flaccidum, and M. polymorpha using the genome databases, and their intracellular localizations and DNA binding activities were investigated by cell biological/biochemical analyses. Based on these results, we propose a model that recurrent modification of cp nucleoid organization by eukaryotic factors originally related to chromatin organization might have been the driving force for the diversification of cp nucleoids since the early stage of green plant evolution.