Light-driven Proton Pumps as a Potential Regulator for Carbon Fixation in Marine Diatoms.

Diatoms are a major phytoplankton group responsible for approximately 20% of carbon fixation on Earth. They perform photosynthesis using light-harvesting chlo-rophylls located in plastids, an organelle obtained through eukaryote-eukaryote endosymbiosis. Microbial rhodopsin, a photoreceptor distinct from chlo-rophyll-based photosystems, was recently identified in some diatoms. However, the physiological function of diatom rhodopsin remains unclear. Heterologous expression techniques were herein used to investigate the protein function and subcellular localization of diatom rhodopsin. We demonstrated that diatom rhodopsin acts as a light-driven proton pump and localizes primarily to the outermost membrane of four membrane-bound complex plastids. Using model simulations, we also examined the effects of pH changes inside the plastid due to rhodopsin-mediated proton transport on photosynthesis. The results obtained suggested the involvement of rhodopsin-mediated local pH changes in a photosynthetic CO2-concentrating mechanism in rhodopsin-possessing diatoms.

Genome evolution of a nonparasitic secondary heterotroph, the diatom Nitzschia putrida.

Secondary loss of photosynthesis is observed across almost all plastid-bearing branches of the eukaryotic tree of life. However, genome-based insights into the transition from a phototroph into a secondary heterotroph have so far only been revealed for parasitic species. Free-living organisms can yield unique insights into the evolutionary consequence of the loss of photosynthesis, as the parasitic lifestyle requires specific adaptations to host environments. Here, we report on the diploid genome of the free-living diatom Nitzschia putrida (35 Mbp), a nonphotosynthetic osmotroph whose photosynthetic relatives contribute ca. 40% of net oceanic primary production. Comparative analyses with photosynthetic diatoms and heterotrophic algae with parasitic lifestyle revealed that a combination of gene loss, the accumulation of genes involved in organic carbon degradation, a unique secretome, and the rapid divergence of conserved gene families involved in cell wall and extracellular metabolism appear to have facilitated the lifestyle of a free-living secondary heterotroph.

An Enigmatic Stramenopile Sheds Light on Early Evolution in Ochrophyta Plastid Organellogenesis.

Ochrophyta is an algal group belonging to the Stramenopiles and comprises diverse lineages of algae which contribute significantly to the oceanic ecosystems as primary producers. However, early evolution of the plastid organelle in Ochrophyta is not fully understood. In this study, we provide a well-supported tree of the Stramenopiles inferred by the large-scale phylogenomic analysis that unveils the eukaryvorous (nonphotosynthetic) protist Actinophrys sol (Actinophryidae) is closely related to Ochrophyta. We used genomic and transcriptomic data generated from A. sol to detect molecular traits of its plastid and we found no evidence of plastid genome and plastid-mediated biosynthesis, consistent with previous ultrastructural studies that did not identify any plastids in Actinophryidae. Moreover, our phylogenetic analyses of particular biosynthetic pathways provide no evidence of a current and past plastid in A. sol. However, we found more than a dozen organellar aminoacyl-tRNA synthases (aaRSs) that are of algal origin. Close relationships between aaRS from A. sol and their ochrophyte homologs document gene transfer of algal genes that happened before the divergence of Actinophryidae and Ochrophyta lineages. We further showed experimentally that organellar aaRSs of A. sol are targeted exclusively to mitochondria, although organellar aaRSs in Ochrophyta are dually targeted to mitochondria and plastids. Together, our findings suggested that the last common ancestor of Actinophryidae and Ochrophyta had not yet completed the establishment of host-plastid partnership as seen in the current Ochrophyta species, but acquired at least certain nuclear-encoded genes for the plastid functions.

Construction of a genetic linkage map and detection of quantitative trait locus for the ergothioneine content in tamogitake mushroom (Pleurotus cornucopiae var. citrinopileatus).

Developing high-content strains of L-ergothioneine (EGT), an antioxidant amino acid, is an important breeding target for tamogitake mushroom, Pleurotus cornucopiae var. citrinopileatus. We constructed a genetic linkage map based on segregation analysis of markers in 105 F1 progenies. The loci of 245 markers, including 10 AFLP markers, 195 Rad markers, 2 mating type factors, and 38 gene markers, were mapped. The map contained 12 linkage groups with a total genetic distance of 906.8 cM, and an average marker interval of 4.0 cM. The population from crossing between tester monokaryon and F1 progenies was used to characterize quantitative trait loci (QTL) for EGT content. With composite interval mapping (CIM) method, QTL of EGT content were found to be located in linkage group 10, having a Logarithm of the odds (LOD) score of 2.53 with a 10.1% contribution rate. Moreover, a single nucleotide polymorphism (SNP), A/T, was identified in a gene region of the genome in the neighborhood where the QTL peak existed. This SNP genotype was in good agreement with the EGT phenotypes of each strain in the both QTL population and wild population. Thus, this SNP would have great potential value to use the marker-assisted selection (MAS) for this mushroom with high EGT content.

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.

Identification of a SNP and development of a PCR-based allele-specific marker of the sporulation-deficient (sporeless) trait of the Tamogitake 108Y2D mutant using next-generation sequencing.

The mass scattering of basidiospores during the cultivation of edible mushrooms causes serious problems, such as allergic reactions in workers. Sporulation-deficient (sporeless) cultivars would be very useful for preventing these issues. We aimed to identify the single-nucleotide polymorphism (SNP) that is responsible for the single dominant sporeless mutation of the Tamogitake 108Y2D mutant using next-generation sequencing (NGS) and TILLING technology and to develop an allele-specific PCR marker for sporeless breeding. By comparing the sequences of the wild-type and its mutant genomes, we identified 685 mutation loci in gene regions and pinpointed one SNP only consistent with sporeless phenotype for 105 segregants, i.e., a C to T located at position 1,950 of the exonic region of a putative fungal transcription factor that generated a stop codon. We developed an allele-specific marker based on the identified SNP, and its high practicality was validated using tests against progenies from several hybrids and wild isolates from different geographical origins. Thus, the allele-specific PCR marker developed here will be useful for marker-assisted selection in the breeding of the sporeless trait of this mushroom. Furthermore, the technical success of SNP identification and marker development based on NGS genome data can help achieve efficient mutation breeding in mushrooms.

Principles of plastid reductive evolution illuminated by nonphotosynthetic chrysophytes.

The division of life into producers and consumers is blurred by evolution. For example, eukaryotic phototrophs can lose the capacity to photosynthesize, although they may retain vestigial plastids that perform other essential cellular functions. Chrysophyte algae have undergone a particularly large number of photosynthesis losses. Here, we present a plastid genome sequence from a nonphotosynthetic chrysophyte, "Spumella" sp. NIES-1846, and show that it has retained a nearly identical set of plastid-encoded functions as apicomplexan parasites. Our transcriptomic analysis of 12 different photosynthetic and nonphotosynthetic chrysophyte lineages reveals remarkable convergence in the functions of these nonphotosynthetic plastids, along with informative lineage-specific retentions and losses. At one extreme, Cornospumella fuschlensis retains many photosynthesis-associated proteins, although it appears to have lost the reductive pentose phosphate pathway and most plastid amino acid metabolism pathways. At the other extreme, Paraphysomonas lacks plastid-targeted proteins associated with gene expression and all metabolic pathways that require plastid-encoded partners, indicating a complete loss of plastid DNA in this genus. Intriguingly, some of the nucleus-encoded proteins that once functioned in the expression of the Paraphysomonas plastid genome have been retained. These proteins were likely to have been dual targeted to the plastid and mitochondria of the chrysophyte ancestor, and are uniquely targeted to the mitochondria in Paraphysomonas Our comparative analyses provide insights into the process of functional reduction in nonphotosynthetic plastids.

Diversity of Organellar Genomes in Non-photosynthetic Diatoms.

We determined the complete sequences of the plastid and mitochondrial genomes of three non-photosynthetic Nitzschia spp., as well as those of a photosynthetic close relative, Nitzschia palea. All the plastid genomes and the three mitochondrial genomes determined were found to be circularly mapping, and the other mitochondrial genomes were predicted to be of a linear form with telomere-like structures at both ends. We found that all the non-photosynthetic plastid genomes are streamlined and lack a common gene set: two RNA genes, and 60 protein-coding genes, most of which are related to photosynthetic functions. Nevertheless, the non-photosynthetic plastid genomes commonly retain ATP synthase complex genes, although atpE is missing in Nitzschia sp. NIES-3581 and three other non-photosynthetic species lack atpF instead of atpE. This observation suggests an evolutionary constraint against the loss of ATP synthase complex genes. All the non-photosynthetic diatom plastid genomes lacked two genes, thiS and thiG, involved in thiamin biosynthesis. Consistent with this gene loss, non-photosynthetic Nitzschia spp. were incapable of thriving in vitamin B1-lacking media. This study clearly demonstrated not only the evolutionary trends of plastid genome reduction but also the linkage between plastid genome reduction and a biological change of nutrient requirements in Nitzschia.

Waste wood recycling as animal bedding and development of bio-monitoring tool using the CALUX assay.

Animal bedding made of waste wood samples from seven different plants in Japan were chemically analyzed in terms of persistent organic pollutants (POPs) including polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/DFs), coplanar polychlorinated biphenyls (Co-PCBs), drin compounds, chlordane compounds and various inorganic toxic compounds (Cr, Cu, As, B, Cd and Pb) to investigate the chemical characteristics and levels of contamination. Further investigation was conducted to determine the success of applying the Chemically Activated Luciferase Expression (CALUX) bioassay to the waste wood samples in combination with a cleanup procedure for the detection of dioxin-like compounds in order to develop the CALUX bioassay as a rapid and cost-effective screening/monitoring method and a contributive tool to risk management in the waste wood recycling process. For the cleanup procedure, crude extracts from wood samples were prepared by dimethylsulfoxide (DMSO)/n-hexane extraction, and then the extracts were processed by silica gel-44% sulfuric acid reflux treatment at 70 degrees C for 60 min to yield the bioassay fractions. The presence of POPs and inorganic toxic compounds were confirmed in most of the litter samples. In particular, Co-PCBs in one sample (litter dust) showed a high concentration level (1200000 pg/g, 240 pg TEQ/g), suggesting the potential for contamination from demolition waste. The CALUX assay-determined TEQs (CALUX-TEQs) were significantly high in the sample after DMSO/n-hexane extraction, probably due to labile aryl hydrocarbon receptor (AhR) ligands such as PAHs; however, they were remarkably reduced through a single silica gel-44% sulfuric acid reflux treatment. The ratio between CALUX-TEQ values and WHO toxicity equivalent values (WHO-TEQ) obtained by congener-specific chemical analysis ranged from 0.058 to 22 and show comparatively good agreement. Underestimation in some samples, however, was observed where WHO-TEQ values of Co-PCBs contributed greatly to total WHO-TEQ values. Reasons for this gap could be lower CALUX assay-determined relative potencies (REPs) than the WHO-TEFs for these congeners or AhR-antagonistic effects of non dioxin-like PCBs which coexist at higher concentration than Co-PCBs. The CALUX assay is proposed as a promising application in the recycling process of wooden materials.