Hiroaki Aoyama

Complete elimination of maternal mitochondrial DNA during meiosis resulting in the paternal inheritance of the mitochondrial genome in Chlamydomonas species

Summary.The non-Mendelian inheritance of organellar DNA is common in most plants and animals. In the isogamous green alga Chlamydomonas species, progeny inherit chloroplast genes from the maternal parent, as paternal chloroplast genes are selectively eliminated in young zygotes. Mitochondrial genes are inherited from the paternal parent. Analogically, maternal mitochondrial DNA (mtDNA) is thought to be selectively eliminated. Nevertheless, it is unclear when this selective elimination occurs. Here, we examined the behaviors of maternal and paternal mtDNAs by various methods during the period between the beginning of zygote formation and zoospore formation. First, we observed the behavior of the organelle nucleoids of living cells by specifically staining DNA with the fluorochrome SYBR Green I and staining mitochondria with 3,3′-dihexyloxacarbocyanine iodide. We also examined the fate of mtDNA of male and female parental origin by real-time PCR, nested PCR with single zygotes, and fluorescence in situ hybridization analysis. The mtDNA of maternal origin was completely eliminated before the first cell nuclear division, probably just before mtDNA synthesis, during meiosis. Therefore, the progeny inherit the remaining paternal mtDNA. We suggest that the complete elimination of maternal mtDNA during meiosis is the primary cause of paternal mitochondrial inheritance.

A quantitative protocol for DNA metabarcoding of springtails (Collembola).

We developed a novel protocol with superior quantitative analysis results for DNA metabarcoding of Collembola, a major soil microarthropod order. Degenerate PCR primers were designed for conserved regions in the mitochondrial cytochrome c oxidase subunit I (mtCOI) and 16S ribosomal RNA (mt16S) genes based on published collembolan mitogenomes. The best primer pair was selected based on its ability to amplify each gene, irrespective of the species. DNA was extracted from 10 natural communities sampled in a temperate forest (with typically 25-30 collembolan species per 10 soil samples) and 10 mock communities (with seven cultured collembolan species). The two gene regions were then amplified using the selected primers, ligated with adapters for 454 technology, and sequenced. Examination of the natural community samples showed that 32 and 36 operational taxonomic units (defined at a 90% sequence similarity threshold) were recovered from the mtCOI and mt16S data, respectively, which were comparable to the results of the microscopic identification of 25 morphospecies. Further, sequence abundances for each collembolan species from the mtCOI and mt16S data of the mock communities, after normalization by using a species as the internal control, showed good correlation with the number of individuals in the samples (R = 0.91-0.99), although relative species abundances within a mock community sample estimated from sequences were skewed from community composition in terms of the number of individuals or biomass of the species. Thus, this protocol enables the comparison of collembolan communities in a quantitative manner by metabarcoding.

The dynamic behaviour of mitochondria in living zygotes during maturation and meiosis in Chlamydomonas reinhardtii

To understand fully the function of mitochondria during the development of cells and organs, it is important to elucidate the dynamics of their morphology. However, the detailed morphology of mitochondria during meiosis has not yet been studied in algae. We examined the mitochondrial morphology of Chlamydomonas reinhardtii and classified zygotes into seven types by mitochondrial morphology in order to analyse the morphological change in mature and meiotic zygotes. We also investigated the oxygen consumption of living zygotes and the effects of tubulin and actin polymerization inhibitors on mitochondria, using fluorescence microscopy and oxygen electrodes. During zygote maturation, mitochondria fragmented into small particles, with a large decrease in oxygen consumption. When mature zygotes were exposed to light, mitochondria became tubular and formed a network, and oxygen consumption gradually recovered. At the same time, particle-like mitochondrial nucleoids became stringy and produced new nucleoid particles. Tubular mitochondria accumulated around the cell nucleus and then spread throughout the cell. Cell division followed (first and second rounds), and the resultant daughter cells had tubular mitochondria in a mesh-like arrangement. An inhibitor of tubulin polymerization, demecolcine, inhibited the assembly of mitochondria around the cell nucleus, whereas an inhibitor of actin polymerization, latrunculin B, inhibited the formation of tubular mitochondria. These results suggest that microtubules are probably involved in mitochondrial accumulation around the cell nucleus, whereas microfilaments may maintain the tubular network of mitochondria.

Comparative analysis of zygospore transcripts during early germination in Chlamydomonas reinhardtii.

The unicellular green alga Chlamydomonas reinhardtii has a haplontic life cycle, and forms diploid zygotes for reproduction. The zygospore, a sporulating zygote, begins germination in response to light signals, generating haploid progenies and inducing several cell-biological events; e.g., DNA synthesis and meiotic division, successively. Their regulatory mechanisms remain largely unknown, so we focused on the early stages of germination and analyzed the dynamics of gene expression associated with the germination process. The gene expression levels of zygospores at 1 and 6h after light exposure were analyzed by a next-generation sequencing platform, the 454 GS Junior. At 6h, the photosynthesis pathway, including its antenna proteins and two methionine metabolism-related genes (methionine synthase and sulfite reductase), were up-regulated compared to 1h after light exposure. Meanwhile, three uncharacterized genes that contained an antibiotic biosynthesis monooxygenase domain and an HSP20/alpha crystallin family protein were specifically expressed at 1h after light exposure. These gene expressions were also verified by quantitative real-time PCR analysis. These results suggest that the photosynthesis and methionine synthesis pathways, both of which occur in the chloroplast, are activated in zygospores at around 6h after light exposure, and that some polyketides and/or a small heat shock protein may be related to the initiation of zygospore germination.

Observations of chromosomal behaviour in living meiotic zygotes of Chlamydomonas reinhardtii (Chlorophyceae)

Clarification of chromosome number and behaviour during meiosis is important for genetic investigations of the unicellular alga Chlamydomonas reinhardtii. However, the chromosome number and their morphology remain uncertain in this alga. We stained living zygotes of C. reinhardtii with the highly sensitive DNA fluorochrome SYBR Green I and observed chromosomal behaviour during meiosis. Zygotes with two and four cells, resulting from the first and second meiotic divisions, appeared approximately 16 and 20 h, respectively, after mature zygotes were exposed to light under experimental conditions. In prophase I, chromosomes in the nucleus changed from long, thread-like structures to bivalents. At metaphase I and anaphase I, chromosomes assumed raft-like configurations but chromosomal configurations were less distinct at metaphase II and anaphase II. The nuclei were always localized near the zygotic surface. By observing bivalents at diakinesis we determined the total number of chromosomes to be 18. Chromosome lengths ranged from 0.5 to 2 µm. Using approximate fluorescence intensities, we estimated DNA content of the largest chromosome to be 3–6 times greater than that of the smallest.

Gene expression analysis of disabled and re-induced isoprene emission by the tropical tree Ficus septica before and after cold ambient temperature exposure.

Isoprene is the most abundant type of nonmethane, biogenic volatile organic compound in the atmosphere, and it is produced mainly by terrestrial plants. The tropical tree species Ficus septica Burm. F. (Rosales: Moraceae) has been shown to cease isoprene emissions when exposed to temperatures of 12 °C or lower and to re-induce isoprene synthesis upon subsequent exposure to temperatures of 30 °C or higher for 24 h. To elucidate the regulation of genes underlying the disabling and then induction of isoprene emission during acclimatization to ambient temperature, we conducted gene expression analyses of F. septica plants under changing temperature using quantitative real-time polymerase chain reaction and western blotting. Transcription levels were analyzed for 17 genes that are involved in metabolic pathways potentially associated with isoprene biosynthesis, including isoprene synthase (ispS). The protein levels of ispS were also measured. Changes in transcription and protein levels of the ispS gene, but not in the other assessed genes, showed identical temporal patterns to isoprene emission capacity under the changing temperature regime. The ispS protein levels strongly and positively correlated with isoprene emission capacity (R(2) = 0.92). These results suggest that transcriptional regulation of ispS gave rise to the temporal variation in isoprene emission capacity in response to changing temperature.

Complete Genome Sequence of the Intracellular Bacterial Symbiont TC1 in the Anaerobic Ciliate Trimyema compressum

ABSTRACT A free-living ciliate, Trimyema compressum, found in anoxic freshwater environments harbors methanogenic archaea and a bacterial symbiont named TC1 in its cytoplasm. Here, we report the complete genome sequence of the TC1 symbiont, consisting of a 1.59-Mb chromosome and a 35.8-kb plasmid, which was determined using the PacBio RSII sequencer.

Predominant expression and activity of vacuolar H(+)-ATPases in the mixed segment of the wood-feeding termite Nasutitermes takasagoensis.

The mixed segment is a unique part of the gut present only in the most apical lineage of termites and consists of a complex of overlapping mesenteric and proctodeal epithelia. In spite of its unique structure, the physiological functions of the mixed segment have been poorly studied. We performed transcriptome analysis to identify functional enzymes acting in the mixed segment of the wood-feeding higher termite Nasutitermes takasagoensis. We sequenced the transcripts (4563 isotigs) of the mixed segment and compared them with those of the midgut (4813 isotigs) and the first proctodeal segment (3629 isotigs). We found that vacuolar H(+)-ATPase (V-ATPase) subunits were predominant in the mixed segment, which was confirmed by RT-qPCR analysis. The V-ATPase activity in these three tissues was in a good agreement with the expression patterns, suggesting that V-ATPase is a prevalent enzyme in the mixed segment of the termites. The results confirmed the proposed role of the mixed segment as a transporting epithelium.

A rapid method of non-destructive DNA extraction from individual springtails (Collembola)

In this study, we describe an easy and rapid method for non-destructive DNA extraction from a single Collembola individual, without dissection, lysis of the specimen, or purification of extracted DNA. We demonstrate that, after a single specimen has been heat-treated in Tris-EDTA (TE) buffer using a standard thermocycler, the solution can be used for PCR amplification of the mitochondrial cytochrome c oxidase subunit 1 (COI) gene region, typically used for DNA barcoding. With this method, the morphological features of Collembola commonly used for species identification are well preserved. This DNA extraction method is preferable for DNA barcoding where the sequencing and preservation of a large number of small and fragile specimens are required.

Development and characterization of 24 microsatellite markers in a eublepharid gecko, Goniurosaurus kuroiwae

The Ryukyu ground geckos, which comprise Goniurosaurus kuroiwae (sensu stricto) and four close relatives in the Ryukyu Archipelago, face conservation threats, and are listed as endangered in the Red List of both IUCN and Japan. We developed 24 microsatellite markers in G. kuroiwae and cross-amplified for another species, G. orientalis. All 24 and 22 loci were successfully amplified and genotyped in G. kuroiwae and G. orientalis, respectively. The number of alleles per locus and expected heterozygosity at polymorphic loci ranged from 1 to 11 and 0.050–0.891, respectively, in G. kuroiwae, and ranged from 1 to 15 and 0.050–0.932, respectively, in G. orientalis.