Complete mitochondrial and chloroplast DNA sequences of the freshwater green microalga Medakamo hakoo.

We report the complete organellar genome sequences of an ultrasmall green alga, Medakamo hakoo strain M-hakoo 311, which has the smallest known nuclear genome in freshwater green algae. Medakamo hakoo has 90.8-kb chloroplast and 36.5-kb mitochondrial genomes containing 80 and 33 putative protein-coding genes, respectively. The mitochondrial genome is the smallest in the Trebouxiophyceae algae studied so far. The GC content of the nuclear genome is 73%, but those of chloroplast and mitochondrial genomes are 41% and 35%, respectively. Codon usages in the organellar genomes have a different tendency from that in the nuclear genome. The organellar genomes have unique characteristics, such as the biased encoding of mitochondrial genes on a single strand and the absence of operon structures in chloroplast ribosomal genes. Medakamo hakoo will be helpful for understanding the evolution of the organellar genome and the regulation of gene expression in chloroplasts and mitochondria.

Genomic analysis of an ultrasmall freshwater green alga, Medakamo hakoo.

Ultrasmall algae have attracted the attention of biologists investigating the basic mechanisms underlying living systems. Their potential as effective organisms for producing useful substances is also of interest in bioindustry. Although genomic information is indispensable for elucidating metabolism and promoting molecular breeding, many ultrasmall algae remain genetically uncharacterized. Here, we present the nuclear genome sequence of an ultrasmall green alga of freshwater habitats, Medakamo hakoo. Evolutionary analyses suggest that this species belongs to a new genus within the class Trebouxiophyceae. Sequencing analyses revealed that its genome, comprising 15.8 Mbp and 7629 genes, is among the smallest known genomes in the Viridiplantae. Its genome has relatively few genes associated with genetic information processing, basal transcription factors, and RNA transport. Comparative analyses revealed that 1263 orthogroups were shared among 15 ultrasmall algae from distinct phylogenetic lineages. The shared gene sets will enable identification of genes essential for algal metabolism and cellular functions.

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.

SQUAMOSA promoter-binding protein-like 7 mediates copper deficiency response in the presence of high nitrogen in Arabidopsis thaliana.

KEY MESSAGE: SQUAMOSA promoter-binding protein-like 7 mediates copper deficiency response in the presence of high nitrogen even with the sufficient level of copper in Arabidopsis thaliana. Under copper (Cu) deficiency, accumulation of mRNA encoding two Cu/Zn superoxide dismutases, CSD1 and CSD2, is downregulated to save Cu for plastocyanin. This downregulation depends on miR398 and is under the control of SQUAMOSA promoter-binding protein-like7 (SPL7). Arabidopsis seedlings are routinely cultured on Murashige and Skoog medium. However, the high nitrogen (N) content of the medium (60 mM) has been shown to induce a similar response to Cu deficiency. The mRNA and protein levels of CSD1 and CSD2 are reduced under high N conditions, even if the Cu concentration in the medium is sufficient (0.1-0.5 µM). In this study, we show that this symptom, similar to the Cu deficiency, occurred in the presence of high N largely depending on SPL7, suggesting that plants actually sensed Cu deficiency. However, a change in N concentration in the medium did not influence the total Cu concentration in shoots or roots. High N did not increase the protein content in leaves but facilitated rapid seedling growth. We speculate that this rapid growth causes a continuous Cu deficiency mainly because of high Cu uptake by mesophyll cells in the leaves. This idea was supported by the observation that plastocyanin did not overaccumulate at the range of 0.1-0.5 µM Cu with 30 mM N. In contrast, in the presence of 5 µM Cu with 30 mM N, plants accumulate more Cu in plastocyanin in the thylakoid lumen, resulting in a slight Cu deficiency in the chloroplast stroma. This process is independent of SPL7.

Morphology, taxonomy and mating-type loci in natural populations of Volvox carteri in Taiwan.

BACKGROUND: Volvox carteri f. nagariensis is a model taxon that has been studied extensively at the cellular and molecular level. The most distinctive morphological attribute of V. carteri f. nagariensis within V. carteri is the production of sexual male spheroids with only a 1:1 ratio of somatic cells to sperm packets or androgonidia (sperm packet initials). However, the morphology of male spheroids of V. carteri f. nagariensis has been examined only in Japanese strains. In addition, V. carteri f. nagariensis has heterothallic sexuality; male and female sexes are determined by the sex-determining chromosomal region or mating-type locus composed of a > 1 Mbp linear chromosome. Fifteen sex-specific genes and many sex-based divergent shared genes (gametologs) are present within this region. Thus far, such genes have not been identified in natural populations of this species. RESULTS: During a recent fieldwork in Taiwan, we encountered natural populations of V. carteri that had not previously been recorded from Taiwan. In total, 33 strains of this species were established from water samples collected in Northern Taiwan. Based on sequences of the internal transcribed spacer 2 region of nuclear ribosomal DNA and the presence of asexual spheroids with up to 16 gonidia, the species was clearly identified as V. carteri f. nagariensis. However, the sexual male spheroids of the Taiwanese strains generally exhibited a 1:1 to > 50:1 ratio of somatic cells to androgonidia. We also investigated the presence or absence of several sex-specific genes and the sex-based divergent genes MAT3m, MAT3f and LEU1Sm. We did not identify recombination or deletion of such genes between the male and female mating-type locus haplotypes in 32 of the 33 strains. In one putative female strain, the female-specific gene HMG1f was not amplified by genomic polymerase chain reaction. When sexually induced, apparently normal female sexual spheroids developed in this strain. CONCLUSIONS: Male spheroids are actually variable within V. carteri f. nagariensis. Therefore, the minimum ratio of somatic cells to androgonidia in male spheroids and the maximum number of gonidia in asexual spheroids may be diagnostic for V. carteri f. nagariensis. HMG1f may not be directly related to the formation of female spheroids in this taxon.

Rediscovery of the species of 'ancestral Volvox': morphology and phylogenetic position of Pleodorina sphaerica (Volvocales, Chlorophyceae) from Thailand.

Pleodorina sphaerica Iyengar was considered to be a phylogenetic link between Volvox and the type species Pleodorina californica Shaw because it has small somatic cells distributed from the anterior to posterior poles in 64- or 128-celled vegetative colonies. However, cultural studies and molecular and ultrastructural data are lacking in P. sphaerica, and this species has not been recorded since 1951. Here, we performed light and electron microscopy and molecular phylogeny of P. sphaerica based on newly established culture strains originating from Thailand. Morphological features of the present Thai species agreed well with those of the previous studies of the Indian material of P. sphaerica and with those of the current concept of the advanced members of the Volvocaceae. The present P. sphaerica strains exhibited homothallic sexuality; male and facultative female colonies developed within a single clonal culture. Chloroplast multigene phylogeny demonstrated that P. sphaerica was sister to two other species of Pleodorina (P. californica and Pleodorina japonica Nozaki) without posterior somatic cells, and these three species of Pleodorina formed a robust clade, which was positioned distally in the large monophyletic group including nine taxa of Volvox sect. Merrillosphaera and Volvox (sect. Janetosphaera) aureus Ehrenberg. Based on the present phylogenetic results, evolutionary losses of posterior somatic cells might have occurred in the ancestor of P. californica and P. japonica. Thus, P. sphaerica might represent an ancestral morphology of Pleodorina, rather than of Volvox.

Primitive red alga Cyanidioschyzon merolae accumulates storage glucan and triacylglycerol under nitrogen depletion.

Most microalgae accumulate neutral lipids, including triacylglycerol (TAG), into spherical structures called lipid bodies (LBs) under environmental stress conditions such as nutrient depletion. In green algae, starch accumulation precedes TAG accumulation, and the starch is thought to be a substrate for TAG synthesis. However, the relationship between TAG synthesis and the starch content in red algae, as well as how TAG accumulation is regulated, is unclear. In this study, we cultured the primitive red alga Cyanidioschyzon merolae under nitrogen-depleted conditions, and monitored the formation of starch granules (SGs) and LBs using microscopy. SGs stained with potassium iodide were observed at 24 h; however, LBs stained specifically with BODIPY 493/503 were observed after 48 h. Quantitative analysis of neutral sugar and cytomorphological semi-quantitative analysis of TAG accumulation also supported these results. Thus, the accumulation of starch occurred and preceded the accumulation of TAG in cells of C. merolae. However, TAG accumulation was not accompanied by a decrease in the starch content, suggesting that the starch is a major carbon storage sink, at least under nitrogen-depleted conditions. Quantitative real-time PCR revealed that the mRNA levels of genes involved in starch and TAG synthesis rarely changed during the culture period, suggesting that starch and TAG synthesis in C. merolae are not controlled through gene transcription but at other stages, such as translation and/or enzymatic 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.

Two possible barriers blocking conjugation between different megakaryotypes of Blepharisma.

We investigated mating pair formation between three Blepharisma species Blepharisma undulans, Blepharisma japonicum, and Blepharisma stoltei to determine whether their respective gamones (mating pheromones) effectively induce mating pairs between different species. Cell-free fluid from type II cells (CFF2) of B. undulans (megakaryotype II) induced pairing of B. japonicum and B. stoltei type I cells (megakaryotype IV), and CFF2 of B. japonicum and B. stoltei induced pairing of B. undulans type I cells. Cell-free fluid from B. undulans type I cells (CFF1) did not induce pairing of B. japonicum and B. stoltei type II cells, and CFF1 of B. japonicum and B. stoltei failed to induce pairing of B. undulans. CFF1 from B. japonicum and B. stoltei mutually induced pairing, as previously reported. These results indicate that gamone 2 is common among megakaryotypes II and IV, and that gamone 1 appears to be at least megakaryotype-specific. When cells belonging to megakaryotypes II and IV are separately pre-treated with effective gamones and mixed, mating pairs between megakaryotypes rarely form. Taken together, these results suggest at least two barriers, a gamone and a factor involved in pair formation, that prevent conjugation between different megakaryotypes of Blepharisma.

Accumulation of TIP2;2 Aquaporin during Dark Adaptation Is Partially PhyA Dependent in Roots of Arabidopsis Seedlings.

Light regulates the expression and function of aquaporins, which are involved in water and solute transport. In Arabidopsis thaliana, mRNA levels of one of the aquaporin genes, TIP2;2, increase during dark adaptation and decrease under far-red light illumination, but the effects of light at the protein level and on the mechanism of light regulation remain unknown. Numerous studies have described the light regulation of aquaporin genes, but none have identified the regulatory mechanisms behind this regulation via specific photoreceptor signaling. In this paper, we focus on the role of phytochrome A (phyA) signaling in the regulation of the TIP2;2 protein. We generated Arabidopsis transgenic plants expressing a TIP2;2-GFP fusion protein driven by its own promoter, and showed several differences in TIP2;2 behavior between wild type and the phyA mutant. Fluorescence of TIP2;2-GFP protein in the endodermis of roots in the wild-type seedlings increased during dark adaptation, but not in the phyA mutant. The amount of the TIP2;2-GFP protein in wild-type seedlings decreased rapidly under far-red light illumination, and a delay in reduction of TIP2;2-GFP was observed in the phyA mutant. Our results imply that phyA, cooperating with other photoreceptors, modulates the level of TIP2;2 in Arabidopsis roots.

Histone H3 is absent from organelle nucleoids in BY-2 cultured tobacco cells.

The core histone proteins (H2A, H2B, H3 and H4) are nuclear-localised proteins that play a central role in the formation of nucleosome structure. They have long been considered to be absent from extra-nuclear, DNA-containing organelles; that is plastids and mitochondria. Recently, however, the targeting of core histone H3 to mitochondria, and the presence of nucleosome-like structures in mitochondrial nucleoids, were proposed in cauliflower and tobacco respectively. Thus, we examined whether histone H3 was present in plant organelles and participated in the organisation of nucleoid structure, using highly purified organelles and organelle nucleoids isolated from BY-2 cultured tobacco cells. Immunofluorescence microscopic observations and Western blotting analyses demonstrated that histone H3 was absent from organelles and organelle nucleoids, consistent with the historical hypothesis. Thus, the organisation of organelle nucleoids, including putative nucleosome-like repetitive structures, should be constructed and maintained without participation of histone H3.

Effects of chloroplast dysfunction on mitochondria: white sectors in variegated leaves have higher mitochondrial DNA levels and lower dark respiration rates than green sectors.

Co-ordination between plastids and mitochondria is so essential that there should be extensive inter-organellar crosstalk during development of plant cells. Indeed, chloroplast dysfunction in white leaves of plastid ribosome-deficient mutant barley, albostrians, is reportedly accompanied by increases in the levels of mitochondrial DNA and mitochondrial transcripts, suggesting that (i) developmental/physiological status of plastids (or heterotrophic growth condition of albino leaves) can affect the status of mitochondrial genome, and (ii) the function of the affected mitochondria may also be up-regulated accordingly. However, functional aspects of the mitochondria affected by chloroplast dysfunction have not yet been examined in detail. Here, we examined the effects of chloroplast dysfunction on mitochondrial DNA level and dark respiration rate, by comparing white and green sectors within individual variegated leaves, using 12 ornamental plants as experimental materials. The pattern of leaf variegation differed from species to species, suggesting that different mechanisms were involved in the formation of white sectors in different species. Quantitative hybridization analysis revealed that mitochondrial DNA levels were generally higher in white sectors than in green sectors. In spite of the elevated mitochondrial DNA levels, however, dark respiration rates in white sectors were generally lower than those in green sectors. Several possible mechanisms for elevation of mitochondrial DNA level and suppression of dark respiration rates in white sectors are discussed.

NtPolI-like1 and NtPolI-like2, bacterial DNA polymerase I homologs isolated from BY-2 cultured tobacco cells, encode DNA polymerases engaged in DNA replication in both plastids and mitochondria.

Two cDNAs encoding homologs of bacterial DNA polymerase I were isolated from cultured tobacco (Nicotiana tabacum) BY-2 cells, and the corresponding genes were named NtPolI-like1 and NtPolI-like2. High sequence similarity suggested that they are orthologous genes each derived from respective parental species of N. tabacum, an allotetraploid plant. Each of the NtPolI-like1/2 gene products had a putative transit peptide for plastid localization at the N-terminus, followed by a 3'-5' exonuclease domain in the internal region, and a DNA polymerase domain in the C-terminal region. Among family A DNA polymerases, NtPolI-like proteins formed, together with other plant DNA polymerase I homologs, a phylogenetic group distinct from mitochondrial DNA polymerase gamma in animals and fungi, as well as eukaryotic cell nuclear-localized repair enzymes. In contrast to computer predictions, experiments with green fluorescent protein (GFP) fusion protein and Western blotting analysis suggested dual targeting of the gene products to both plastids and mitochondria. The recombinant NtPolI-like2 protein exhibited DNA polymerase activity in vitro. Their biochemical character roughly coincided with those of the 116 kDa DNA polymerases found in the plastid and mitochondrial nuclei (nucleoids) isolated from BY-2 cells. Pre-treatment of the organelle nuclear extracts with anti-NtPolI-like antibody removed most of the DNA polymerase activity. Reverse transcription-PCR (RT-PCR) and Western blotting analyses demonstrated transient activation of NtPolI-like gene expression in the initial phase of cell proliferation, exactly when the 116 kDa DNA polymerases in the isolated organelle nuclei were activated and preferential synthesis of organelle DNAs occurred. Taken together, our results suggest that NtPolI-like1/2 genes encode DNA polymerases engaged in DNA replication in both plastids and mitochondria.