Madis Metsis

Large‐scale parallel 454 sequencing reveals host ecological group specificity of arbuscular mycorrhizal fungi in a boreonemoral forest

* Knowledge of the diversity of arbuscular mycorrhizal fungi (AMF) in natural ecosystems is a major bottleneck in mycorrhizal ecology. Here, we aimed to apply 454 sequencing--providing a new level of descriptive power--to assess the AMF diversity in a boreonemoral forest. * 454 sequencing reads of the small subunit ribosomal RNA (SSU rRNA) gene of Glomeromycota were assigned to sequence groups by blast searches against a custom-made annotated sequence database. * We detected 47 AMF taxa in the roots of 10 plant species in a 10 x 10 m plot, which is almost the same as the number of plant species in the whole studied forest. There was a significant difference between AMF communities in the roots of forest specialist plant species and in the roots of habitat generalist plant species. Forest plant species hosted 22 specialist AMF taxa, and the generalist plants shared all but one AMF taxon with forest plants, including globally distributed generalist fungi. These AMF taxa that have been globally recorded only in forest ecosystems were significantly over-represented in the roots of forest plant species. * Our findings suggest that partner specificity in AM symbiosis may occur at the level of ecological groups, rather than at the species level, of both plant and fungal partners.

Global sampling of plant roots expands the described molecular diversity of arbuscular mycorrhizal fungi.

We aimed to enhance understanding of the molecular diversity of arbuscular mycorrhizal fungi (AMF) by building a new global dataset targeting previously unstudied geographical areas. In total, we sampled 96 plant species from 25 sites that encompassed all continents except Antarctica. AMF in plant roots were detected by sequencing the nuclear SSU rRNA gene fragment using either cloning followed by Sanger sequencing or 454-sequencing. A total of 204 AMF phylogroups (virtual taxa, VT) were recorded, increasing the described number of Glomeromycota VT from 308 to 341 globally. Novel VT were detected from 21 sites; three novel but nevertheless widespread VT (Glomus spp. MO-G52, MO-G53, MO-G57) were recorded from six continents. The largest increases in regional VT number were recorded in previously little-studied Oceania and in the boreal and polar climatic zones — this study providing the first molecular data from the latter. Ordination revealed differences in AM fungal communities between different continents and climatic zones, suggesting that both biogeographic history and environmental conditions underlie the global variation of those communities. Our results show that a considerable proportion of Glomeromycota diversity has been recorded in many regions, though further large increases in richness can be expected in remaining unstudied areas.

Species richness of arbuscular mycorrhizal fungi: associations with grassland plant richness and biomass

Although experiments show a positive association between vascular plant and arbuscular mycorrhizal fungal (AMF) species richness, evidence from natural ecosystems is scarce. Furthermore, there is little knowledge about how AMF richness varies with belowground plant richness and biomass. We examined relationships among AMF richness, above- and belowground plant richness, and plant root and shoot biomass in a native North American grassland. Root-colonizing AMF richness and belowground plant richness were detected from the same bulk root samples by 454-sequencing of the AMF SSU rRNA and plant trnL genes. In total we detected 63 AMF taxa. Plant richness was 1.5 times greater belowground than aboveground. AMF richness was significantly positively correlated with plant species richness, and more strongly with below- than aboveground plant richness. Belowground plant richness was positively correlated with belowground plant biomass and total plant biomass, whereas aboveground plant richness was positively correlated only with belowground plant biomass. By contrast, AMF richness was negatively correlated with belowground and total plant biomass. Our results indicate that AMF richness and plant belowground richness are more strongly related with each other and with plant community biomass than with the plant aboveground richness measures that have been almost exclusively considered to date.

Characterization of the Vaginal Micro- and Mycobiome in Asymptomatic Reproductive-Age Estonian Women

The application of high-throughput sequencing methods has raised doubt in the concept of the uniform healthy vaginal microbiota consisting predominantly of lactobacilli by revealing the existence of more variable bacterial community composition. As this needs to be analyzed more extensively and there is little straightforward data regarding the vaginal mycobiome of asymptomatic women we aimed to define bacterial and fungal communities in vaginal samples from 494 asymptomatic, reproductive-age Estonian women. The composition of the vaginal microbiota was determined by amplifying bacterial 16S rRNA and fungal internal transcribed spacer-1 (ITS-1) regions and subsequently sequencing them using 454 Life Sciences pyrosequencing. We delineated five major bacterial community groups with distinctive diversity and species composition. Lactobacilli were among the most abundant bacteria in all groups, but also members of genus Gardnerella had high relative abundance in some of the groups. Microbial diversity increased with higher vaginal pH values, and was also higher when a malodorous discharge was present, indicating that some of the women who consider themselves healthy may potentially have asymptomatic bacterial vaginosis (BV). Our study is the first of its kind to analyze the mycobiome that colonizes the healthy vaginal environment using barcoded pyrosequencing technology. We observed 196 fungal operational taxonomic units (OTUs), including 16 OTUs of Candida spp., which is more diverse than previously recognized. However, assessing true fungal diversity was complicated because of the problems regarding the possible air-borne contamination and bioinformatics used for identification of fungal taxons as significant proportion of fungal sequences were assigned to unspecified OTUs.

Plant species richness belowground: higher richness and new patterns revealed by next-generation sequencing

Variation in plant species richness has been described using only aboveground vegetation. The species richness of roots and rhizomes has never been compared with aboveground richness in natural plant communities. We made direct comparisons of grassland plant richness in identical volumes (0.1 × 0.1 × 0.1 m) above and below the soil surface, using conventional species identification to measure aboveground richness and 454 sequencing of the chloroplast trnL(UAA) intron to measure belowground richness. We described above‐ and belowground richness at multiple spatial scales (from a neighbourhood scale of centimetres to a community scale of hundreds of metres), and related variation in richness to soil fertility. Tests using reference material indicated that 454 sequencing captured patterns of species composition and abundance with acceptable accuracy. At neighbourhood scales, belowground richness was up to two times greater than aboveground richness. The relationship between above‐ and belowground richness was significantly different from linear: beyond a certain level of belowground richness, aboveground richness did not increase further. Belowground richness also exceeded that of aboveground at the community scale, indicating that some species are temporarily dormant and absent aboveground. Similar to other grassland studies, aboveground richness declined with increasing soil fertility; in contrast, the number of species found only belowground increased significantly with fertility. These results indicate that conventional aboveground studies of plant richness may overlook many coexisting species, and that belowground richness becomes relatively more important in conditions where aboveground richness decreases. Measuring plant belowground richness can considerably alter perceptions of biodiversity and its responses to natural and anthropogenic factors.

Anthropogenic land use shapes the composition and phylogenetic structure of soil arbuscular mycorrhizal fungal communities

Arbuscular mycorrhizal (AM) fungi play an important role in ecosystems, but little is known about how soil AM fungal community composition varies in relation to habitat type and land-use intensity. We molecularly characterized AM fungal communities in soil samples (n = 88) from structurally open (permanent grassland, intensive and sustainable agriculture) and forested habitats (primeval forest and spruce plantation). The habitats harboured significantly different AM fungal communities, and there was a broad difference in fungal community composition between forested and open habitats, the latter being characterized by higher average AM fungal richness. Within both open and forest habitats, intensive land use significantly influenced community composition. There was a broad difference in the phylogenetic structure of AM fungal communities between mechanically disturbed and nondisturbed habitats. Taxa from Glomeraceae served as indicator species for the nondisturbed habitats, while taxa from Archaeosporaceae, Claroideoglomeraceae and Diversisporaceae were indicators for the disturbed habitats. The distribution of these indicator taxa among habitat types in the MaarjAM global database of AM fungal diversity was in accordance with their local indicator status.

Research Resource: Small RNA-seq of Human Granulosa Cells Reveals miRNAs in FSHR and Aromatase Genes

The granulosa cells in the mammalian ovarian follicle respond to gonadotropin signaling and are involved in the processes of folliculogenesis and oocyte maturation. Studies on gene expression and regulation in human granulosa cells are of interest due to their potential for estimating the oocyte viability and in vitro fertilization success. However, the posttranscriptional gene expression studies on micro-RNA (miRNA) level in the human ovary have been scarce. The current study determined the miRNA profile by deep sequencing of the 2 intrafollicular somatic cell types: mural and cumulus granulosa cells (MGCs and CGCs, respectively) isolated from women undergoing controlled ovarian stimulation and in vitro fertilization. Altogether, 936 annotated and 9 novel miRNAs were identified. Ninety of the annotated miRNAs were differentially expressed between MGCs and CGCs. Bioinformatic prediction revealed that TGFβ, ErbB signaling, and heparan sulfate biosynthesis were targeted by miRNAs in both granulosa cell populations, whereas extracellular matrix remodeling, Wnt, and neurotrophin signaling pathways were enriched among miRNA targets in MGCs. Two of the nine novel miRNAs found were of intronic origin: one from the aromatase and the other from the FSH receptor gene. The latter miRNA was predicted to target the activin signaling pathway. In addition to revealing the genome-wide miRNA signature in human granulosa cells, our results suggest that posttranscriptional regulation of gene expression by miRNAs could play an important role in the modification of gonadotropin signaling. miRNA expression studies could therefore lead to new prognostic markers in assisted reproductive technologies.

Role of mitochondria–cytoskeleton interactions in respiration regulation and mitochondrial organization in striated muscles

The aim of this work was to study the regulation of respiration and energy fluxes in permeabilized oxidative and glycolytic skeletal muscle fibers, focusing also on the role of cytoskeletal protein tubulin βII isotype in mitochondrial metabolism and organization. By analyzing accessibility of mitochondrial ADP, using respirometry and pyruvate kinase-phosphoenolpyruvate trapping system for ADP, we show that the apparent affinity of respiration for ADP can be directly linked to the permeability of the mitochondrial outer membrane (MOM). Previous studies have shown that MOM permeability in cardiomyocytes can be regulated by VDAC interaction with cytoskeletal protein, βII tubulin. We found that in oxidative soleus skeletal muscle the high apparent Km for ADP is associated with low MOM permeability and high expression of non-polymerized βII tubulin. Very low expression of non-polymerized form of βII tubulin in glycolytic muscles is associated with high MOM permeability for adenine nucleotides (low apparent Km for ADP).

Changes in the Transcriptome of the Human Endometrial Ishikawa Cancer Cell Line Induced by Estrogen, Progesterone, Tamoxifen, and Mifepristone (RU486) as Detected by RNA-Sequencing

Background Estrogen (E2) and progesterone (P4) are key players in the maturation of the human endometrium. The corresponding steroid hormone modulators, tamoxifen (TAM) and mifepristone (RU486) are widely used in breast cancer therapy and for contraception purposes, respectively. Methodology/Principal findings Gene expression profiling of the human endometrial Ishikawa cancer cell line treated with E2 and P4 for 3 h and 12 h, and TAM and RU486 for 12 h, was performed using RNA-sequencing. High levels of mRNA were detected for genes, including PSAP, ATP5G2, ATP5H, and GNB2L1 following E2 or P4 treatment. A total of 82 biomarkers for endometrial biology were identified among E2 induced genes, and 93 among P4 responsive genes. Identified biomarkers included: EZH2, MDK, MUC1, SLIT2, and IL6ST, which are genes previously associated with endometrial receptivity. Moreover, 98.8% and 98.6% of E2 and P4 responsive genes in Ishikawa cells, respectively, were also detected in two human mid-secretory endometrial biopsy samples. TAM treatment exhibited both antagonistic and agonistic effects of E2, and also regulated a subset of genes independently. The cell cycle regulator cyclin D1 (CCND1) showed significant up-regulation following treatment with TAM. RU486 did not appear to act as a pure antagonist of P4 and a functional analysis of RU486 response identified genes related to adhesion and apoptosis, including down-regulated genes associated with cell-cell contacts and adhesion as CTNND1, JUP, CDH2, IQGAP1, and COL2A1. Conclusions Significant changes in gene expression by the Ishikawa cell line were detected after treatments with E2, P4, TAM, and RU486. These transcriptome data provide valuable insight into potential biomarkers related to endometrial receptivity, and also facilitate an understanding of the molecular changes that take place in the endometrium in the early stages of breast cancer treatment and contraception usage.

Identification of the gene transcription repressor domain of Gli3

Gli transcription factors are downstream targets of the Hedgehog signaling pathway. Two of the three Gli proteins harbor gene transcription repressor function in the N‐terminal half. We have analyzed the sequences and identified a potential repressor domain in Gli2 and Gli3 and have tested this experimentally. Overexpression studies confirm that the N‐terminal parts harbor gene repression activity and we mapped the minimal repressor to residues 106 till 236 in Gli3. Unlike other mechanisms that inhibit Gli induced gene transcription, the repressor domain identified here does not utilize Histone deacetylases (HDACs) to achieve repression, as confirmed by HDAC inhibition studies and pull‐down assays. This distinguishes the identified domain from other regulatory parts with negative influence on transcription.