Mapping Human Transient Transcriptomes Using Single Nucleotide Resolution 4sU Sequencing (SNU-Seq)

Genomes are pervasively transcribed leading to stable and unstable transcripts that define functional regions of genomes and contribute to cellular phenotypes. Defining comprehensive nascent transcriptomes is pivotal to understand gene regulation, disease processes, and the impact of extracellular signals on cells. However, currently employed methods are laborious, technically challenging and costly. We developed single-nucleotide resolution 4sU-sequencing (SNU-Seq), involving pulse labelling, biotinylation and direct isolation of nascent transcripts. Artificial poly-(A)-tailing of the 3' most nucleotide of nascent transcripts ensures oligo-d(T) primer-based library preparation and sequencing using commercial 3' RNA-Seq kits. We show that SNU-Seq is a cost-effective new method generating even read profiles across transcription units. We used SNU-Seq to identify transcription elongation parameters, to map usage of polyadenylation (PAS) sites and novel enhancers. Remarkably, 4sU labelled nascent RNA accumulates short ~100nt transcripts that map to the 5' end of genes. We show that isolation of these short nascent RNA and sequencing the 5' and 3' ends using size-selected SNU-Seq (ssSNU-Seq) provides highly sensitive annotations of mapped and novel TSSs, promoter-proximal pause/termination sites. Thus, SNU-seq and ssSNU-seq combined yield comprehensive transcriptomics data at low cost with high spatial and temporal resolution.

Straw incorporation in deep soil layer promotes net photosynthetic carbon assimilation and maize growth in Northeast China

Background and aims Returning straw into soil could increase soil organic carbon (SOC) and promote crop growth. However, little has been reported on the source of C for increased SOC (straw C or crop photosynthetic C). Methods To investigate the assimilation of photosynthetic C and its distribution in soil in the maize growth season, we set up a one-year 13C pulse-labelling experiment in a consecutive maize straw returning long-term trial. Four treatments were included: no straw return (control), straw mulching on the soil surface (cover), return in 0–20 cm layer (shallow) and 20–40 cm layer (deep). Results We found that the deep straw incorporation significantly (P < 0.05) increased maize grain yield (by 2.9%) and SOC (by 13.4%). During the growing season, the deep straw incorporation increased photosynthetic 13C assimilation in shoots by 17.4% and the partitioning of photosynthetic 13C to soil by 7.9% at early jointing, and by 11.5% at maturity. The contribution of photosynthetic C to microbial biomass C (MBC) and dissolved organic C (DOC) was highest at jointing, and at harvest amounted to 39.1 % of MBC and 28.8% of DOC. Conclusion The results highlighted the importance of regulating the soil carbon dynamics via the deep straw return strategy. In conclusion, deep straw incorporation significantly increased photosynthetic efficiency and facilitated partitioning of photosynthetic C to roots and soil, thus promoting maize growth.

High content genome-wide siRNA screen to investigate the coordination of cell size and RNA production

Coordination of RNA abundance and production rate with cell size has been observed in diverse organisms and cell populations. However, how cells achieve such ‘scaling’ of transcription with size is unknown. Here we describe a genome-wide siRNA screen to identify regulators of global RNA production rates in HeLa cells. We quantify the single-cell RNA production rate using metabolic pulse-labelling of RNA and subsequent high-content imaging. Our quantitative, single-cell measurements of DNA, nascent RNA, proliferating cell nuclear antigen (PCNA), and total protein, as well as cell morphology and population-context, capture a detailed cellular phenotype. This allows us to account for changes in cell size and cell-cycle distribution (G1/S/G2) in perturbation conditions, which indirectly affect global RNA production. We also take advantage of the subcellular information to distinguish between nascent RNA localised in the nucleolus and nucleoplasm, to approximate ribosomal and non-ribosomal RNA contributions to perturbation phenotypes. Perturbations uncovered through this screen provide a resource for exploring the mechanisms of regulation of global RNA metabolism and its coordination with cellular states.

Biomass allocation and seasonal non-structural carbohydrate dynamics do not explain the success of tall forbs in short alpine grassland

The majority of alpine plants are of small stature. Through their small size alpine plants are decoupled from the free atmospheric circulation and accumulate solar heat. However, a few alpine species do not follow that “rule” and protrude with their aboveground structures from the microclimatic shelter of the main canopy boundary layer. We aim at explaining the phenomenon of being tall by exploring the biomass production and carbon relations of four pairs of small and tall phylogenetically related taxa in alpine grassland. We compared species and stature-specific biomass allocation, shifts in non-structural carbohydrate (NSC) concentrations in different tissues throughout the season, and we used 13C labels to track carbon transfer from leaves to belowground structures. Small and tall herbs did not differ in their above- to belowground biomass allocation. The NSC composition (starch, fructan, simple sugars) and allocation did not show a stature-specific pattern, except for higher concentrations of simple sugars in tall species during their extended shoot growth. In relative terms, tall species had higher NSC pools in rhizomes, whereas small species had higher NSC pools in roots. Our findings do not place tall alpine forbs in an exceptional category in terms of biomass allocation and carbohydrate storage. The tall versus small stature of the examined herbs does not seem to be associated with specific adjustments in carbon relations. 13C pulse labelling revealed early C autonomy in young, unfolding leaves of the tall species, which are thus independent of the carbon reserves in the massive belowground organs.

Fluid-phase and membrane markers reveal spatio-temporal dynamics of membrane traffic and repair in the green alga Chara australis

We investigated the mechanisms and the spatio-temporal dynamics of fluid-phase and membrane internalization in the green algaChara australisusing fluorescent hydrazides markers alone, or in conjunction with styryl dyes.Using live-cell imaging, immunofluorescence and inhibitor studies we revealed that both fluid-phase and membrane dyes were actively taken up into the cytoplasm by clathrin-mediated endocytosis and stained various classes of endosomes including brefeldin A- and wortmannin-sensitive organelles (trans-Golgi network and multivesicular bodies). Uptake of fluorescent hydrazides was poorly sensitive to cytochalasin D, suggesting that actin plays a minor role in constitutive endocytosis inCharainternodal cells. Sequential pulse-labelling experiments revealed novel aspects of the temporal progression of endosomes inCharainternodal cells. The internalized fluid-phase marker distributed to early compartments within 10 min from dye exposure and after about 30 min, it was found almost exclusively in late endocytic compartments. Notably, fluid cargo consecutively internalized at time intervals of more than 1h, was not targeted to the same vesicular structures, but was sorted into distinct late compartments. We further found that fluorescent hydrazide dyes distributed not only to rapidly recycling endosomes but also to long-lived compartments that participated in plasma membrane repair after local laser injury. Our approach highlights the benefits of combining different fluid-phase markers in conjunction with membrane dyes in simultaneous and sequential application modus for investigating vesicle traffic, especially in organisms, which are still refractory to genetic transformation like characean algae.

Data-informed trait-based modeling of microbial carbon cycling in soil

&lt;p&gt;Soil microbial functional traits control carbon (C) decomposition and stabilization in soil. Integrating metabolic trade-offs and life-history strategies of microbial communities into models enhances the representation of feedbacks between microbial diversity and soil biogeochemical functions. This has great potential to improve our understanding of microbial C allocation and how microbial processes affect C storage and use efficiency in soil. The current challenge is, however, to quantify and identify ecologically meaningful microbial traits. This study utilizes data from a &lt;sup&gt;13&lt;/sup&gt;C pulse-labelling litter decomposition experiment to inform a new soil C turnover model that captures microbial life-history traits and dormancy in combination with soil organic matter accessibility. Quantitative data from &lt;sup&gt;13&lt;/sup&gt;C DNA stable isotope probing and high-throughput sequencing is used to parameterize the C utilization of copiotrophic and oligotrophic microorganisms. The new model is then applied to quantify C utilization of functional microbial groups and C turnover in soil.&amp;#160; In scenario analyses we investigate the sensitivity of functional microbial groups and its feedback on C cycling to C input.&lt;/p&gt;

Temporal patterns of carbon flow from grassland vegetation to soil microorganisms measured using 13C-labelling and signature fatty acids

Purpose We investigated how the C flow from plants to microorganisms varies throughout the year in a temperate grassland. Additionally, we investigated how the C flow relates to saprotrophic activity and vegetation changes. Methods In situ stable isotope pulse labelling (13CO2) was employed to estimate the flow of recently plant-derived C to soil microorganisms by using signature fatty acids. Bacterial and fungal growth was estimated using radio-labelling in laboratory incubations. Results The C flow from plants to arbuscular mycorrhizal (AM) fungi peaked during the warmer parts of the year, but saprotrophic microorganisms showed little temporal variation in C flow. Also saprotrophic fungi received considerable amounts of C from plants throughout the year. Bacterial and fungal growth showed temporal variation with a growth peak in August for both. This suggests a shift in the C source from mainly rhizosphere C in colder parts of the year, to older C-sources in warmer parts of the year (August). Conclusion We conclude that AM fungi, saprotrophic fungi and bacteria differ in the amount of recently-fixed C they receive from plants throughout the year. Hence, temporal patterns need to be considered to understand ecosystem functioning. The studied plant community included winter annuals, which potentially maintain a high C flow to saprotrophic fungi during the cold season.