Chase Period Research Articles

Early changes in carbon uptake and partitioning moderate belowground carbon storage in a perennial grain

There is increasing interest in perennial crops to build soil carbon (C), but the mechanisms underlying soil C accrual in perennial croplands remain unclear, especially over time in the first years of perennial crop growth. To address this gap, research is needed that directly tracks intra-annual C fluxes through crop-microbial-soil pools, evaluating the capacity of perennial crops to build soil C over intra-decadal time periods. We conducted a 13C isotope-tracer study to compare within-season C uptake and crop-microbial-soil C partitioning patterns between 1-year-old (IWG-1) and 2-year-old (IWG-2) stands of a novel perennial grain crop, intermediate wheatgrass (IWG; Thinopyrum intermedium (Host) Barkworth and Dewey). We compared these to a common annual grain crop, spring wheat (Triticum aestivum L.). Crop shoots, roots, soil, and soil respired-C were sampled ten times over a 90-day chase period. We also measured the incorporation of recently assimilated 13C into soil microbial biomass (13C PLFA) and functional groups over the first 7 days post-label application. Overall, IWG-1 assimilated almost 1670 mg 13C m−2 during the study period, nearly twice that of IWG-2 or wheat, but neither IWG system retained significant amounts of new C in soil. Rather, a higher proportion of assimilated new C was retained in IWG-1 in root tissues (14%) and arbuscular mycorrhizal fungi when compared to other cropping systems, while IWG-2 retained almost 50% of total assimilated C in aboveground crop tissues. We expect the shift from new C retention in belowground root-mycorrhizal networks to aboveground tissues is associated with a shift from an acquisitive to conservative growth strategy that occurs between the first and second IWG production years. The observed shift in C partitioning patterns and potential change in growth strategy limited the allocation and retention of new C in soil as IWG aged, adding valuable context to our understanding of why perennial grain crop establishment seldom leads to significant carbon gains in the first several years following establishment.

Leaf day respiration involves multiple carbon sources and depends on previous dark metabolism.

Day respiration (Rd) is the metabolic, nonphotorespiratory process by which illuminated leaves liberate CO2 during photosynthesis. Rd is used routinely in photosynthetic models and is thus critical for calculations. However, metabolic details associated with Rd are poorly known, and this can be problematic to predict how Rd changes with environmental conditions and relates to night respiration. It is often assumed that day respiratory CO2 release just reflects 'ordinary' catabolism (glycolysis and Krebs 'cycle'). Here, we carried out a pulse-chase experiment, whereby a 13CO2 pulse in the light was followed by a chase period in darkness and then in the light. We took advantage of nontargeted, isotope-assisted metabolomics to determine non-'ordinary' metabolism, detect carbon remobilisationand compare light and dark 13C utilisation. We found that several concurrent metabolic pathways ('ordinary' catabolism, oxidative pentose phosphates pathway, amino acid production, nucleotide biosynthesisand secondary metabolism) took place in the light and participated in net CO2 efflux associated with day respiration. Flux reconstruction from metabolomics leads to an underestimation of Rd, further suggesting the contribution of a variety of CO2-evolving processes. Also, the cornerstone of the Krebs 'cycle', citrate, is synthetised de novo from photosynthates mostly in darkness, and remobilised or synthesised from stored material in the light. Collectively, our data provides direct evidence that leaf day respiration (i) involves several CO2-producing reactions and (ii) is fed by different carbon sources, including stored carbon disconnected from current photosynthates.

Role of Dgat2 in Glucose Uptake and Fatty Acid Metabolism in C2C12 Skeletal Myotubes.

Acyl-coenzyme A (CoA):diacylglycerol acyltransferase 2 (DGAT2) catalyzes the last stage of triacylglycerol (TAG) synthesis, a process that forms ester bonds with diacylglycerols (DAG) and fatty acyl-CoA substrates. The enzymatic role of Dgat2 has been studied in various biological species. Still, the full description of how Dgat2 channels fatty acids in skeletal myocytes and the consequence thereof in glucose uptake have yet to be well established. Therefore, this study explored the mediating role of Dgat2 in glucose uptake and fatty acid partitioning under short interfering ribonucleic acid (siRNA)-mediated Dgat2 knockdown conditions. Cells transfected with Dgat2 siRNA downregulated glucose transporter type 4 (Glut4) messenger RNA (mRNA) expression and decreased the cellular uptake of [1-14C]-labeled 2-deoxyglucose up to 24.3% (p < 0.05). Suppression of Dgat2 deteriorated insulininduced Akt phosphorylation. Dgat2 siRNA reduced [1-14C]-labeled oleic acid incorporation into TAG, but increased the level of [1-14C]-labeled free fatty acids at 3 h after initial fatty acid loading. In an experiment of chasing radioisotope-labeled fatty acids, Dgat2 suppression augmented the level of cellular free fatty acids. It decreased the level of re-esterification of free fatty acids to TAG by 67.6% during the chase period, and the remaining pulses of phospholipids and cholesteryl esters were decreased by 34.5% and 61%, respectively. Incorporating labeled fatty acids into beta-oxidation products increased in Dgat2 siRNA transfected cells without gene expression involving fatty acid oxidation. These results indicate that Dgat2 has regulatory function in glucose uptake, possibly through the reaction of TAG with endogenously released or recycled fatty acids.

Composition and contribution of Stipa bungeana root exudates to soil organic carbon fractions in the Loess Plateau

Root exudates play an important role in carbon sequestration in grassland, but the contribution of root exudates to soil organic carbon (SOC) fractions remains unclear. In this study, Stipa bungeana was analyzed, determined the composition and content of root exudates by GC–MS in a soil culture experiment and labeled with 13CO2 in a rhizibox experiment. Esters accounted for 65.38 % of root exudates, among which 1-propen-1, 2, 3-tricarboxylic acid-tributyl ester, and acetyl-tributyl citrate were the main components. During a 65-day chase period, the contents of SOC and its fractions in the rhizosphere were higher than those in bulk soil. After first day of labeling, the SOC concentration decreased by 16.95 % in the rhizosphere and reached the minimum level. Except for 13C-HC in bulk soil, which was detected three days after labeling, the rest were detected 2 h after labeling. In the rhizosphere soil, the amount of 13C in MBC and HC peaked after one day of labeling. At 65 days after labeling, the 13C-DOC, 13C-HC, and 13C-MBC in the rhizosphere significantly decreased by 59.79 %, 75.99 %, and 55.56 %, respectively. Root exudates had a rapid effect on active organic carbon fractions (MBC and DOC), and also affect the humus carbon (HC) in the short term. This study reveals the mechanism of root exudates on SOC pool during plant growth.

Fgfr2b signaling is essential for the maintenance of the alveolar epithelial type 2 lineage during lung homeostasis in mice

Fibroblast growth factor receptor 2b (Fgfr2b) signaling is essential throughout lung development to form the alveolar epithelial lineage. However, its role in alveolar epithelial type 2 cells (AT2s) homeostasis was recently considered dispensable. SftpcCreERT2; Fgfr2bflox/flox; tdTomatoflox/flox mice were used to delete Fgfr2b expression in cells belonging to the AT2 lineage, which contains mature AT2s and a novel SftpcLow lineage-traced population called “injury activated alveolar progenitors” or IAAPs. Upon continuous tamoxifen exposure for either 1 or 2 weeks to delete Fgfr2b, a shrinking of the AT2 population is observed. Mature AT2s exit the cell cycle, undergo apoptosis and fail to form alveolospheres in vitro. However, the lung morphometry appears normal, suggesting the involvement of compensatory mechanisms. In mutant lungs, IAAPs which escaped Fgfr2b deletion expand, display enhanced alveolosphere formation in vitro and increase drastically their AT2 signature, suggesting differentiation towards mature AT2s. Interestingly, a significant increase in AT2s and decrease in IAPPs occurs after a 1-week tamoxifen exposure followed by an 8-week chase period. Although mature AT2s partially recover their alveolosphere formation capabilities, the IAAPs no longer display this property. Single-cell RNA seq analysis confirms that AT2s and IAAPs represent stable and distinct cell populations and recapitulate some of their characteristics observed in vivo. Our results underscore the essential role played by Fgfr2b signaling in the maintenance of the AT2 lineage in the adult lung during homeostasis and suggest that the IAAPs could represent a new population of AT2 progenitors.

Dpp4+ interstitial progenitor cells contribute to basal and high fat diet-induced adipogenesis

ObjectiveThe capacity to generate new adipocytes from precursor cells is critical for maintaining metabolic health. Adipocyte precursor cells (APCs) constitute a heterogenous collection of cell types; however, the contribution of these various cell types to adipose tissue expansion in vivo remains unknown. The aim of the current study is to investigate the contribution of Dpp4+ progenitors to de novo adipogenesis. MethodsSingle cell analysis has identified several transcriptionally distinct subpopulations of APCs, including Dpp4+ progenitor cells concentrated in the connective tissue surrounding many organs, including white adipose tissue (WAT). Here, we generated a Dpp4CreER mouse model for in vivo lineage tracing of these cells and their downstream progeny in the setting of basal or high fat diet (HFD)-stimulated adipogenesis. ResultsDpp4CreER mice enabled specific temporal labeling of Dpp4+ progenitor cells within their native connective tissue niche. Following a dietary chase period consisting of chow or HFD feeding for 18 weeks, Dpp4+ progenitors differentiated into mature adipocytes within the gonadal and subcutaneous WAT. HFD stimulated adipogenic contribution from Dpp4+ cells in the gonadal but not the subcutaneous depot. Flow cytometry analysis revealed that Dpp4+ progenitors give rise to DPP4(−)/ICAM1+ preadipocytes in vivo. HFD feeding did not perturb the flux of Dpp4+ cell conversion into ICAM1+ preadipocytes in gonadal WAT. Conversely, in subcutaneous WAT, HFD feeding/obesity led to an accumulation of ICAM1+ preadipocytes without a corresponding increase in mature adipocyte differentiation. Examination of non-classical murine visceral depots with relevance to humans, including omentum and retroperitoneal WAT, revealed robust contribution of Dpp4+ progenitors to de novo adipogenesis, which was further stimulated by HFD. ConclusionOur data demonstrate that Dpp4+ interstitial progenitor cells contribute to basal adipogenesis in all fat depots and are recruited to support de novo adipogenic expansion of visceral WAT in the setting of HFD-induced obesity.

Biosynthesis of α-solanine and α-chaconine in potato leaves (Solanum tuberosum L.) – A 13CO2 study

α-Solanine and α-chaconine are the major glycoalkaloids (SGAs) in potatoes, but up to now the biosynthesis of these saponins is not fully understood. In planta13CO2 labeling experiments monitored by nuclear magnetic resonance spectroscopy (NMR) and high-resolution mass spectrometry (HRMS) unraveled the SGA biosynthetic pathways from CO2 photosynthates via early precursors to the SGAs. After a pulse of ~ 700 ppm 13CO2 for four hours, followed by a chase period for seven days, specific 13C-distributions were detected in SGAs from the leaves of the labeled plant. NMR analysis determined the positional 13C-enrichments in α-solanine and α-chaconine characterized by 13C2–pairs in their aglycones. These patterns were in perfect agreement with a mevalonate-dependent biosynthesis of the isopentenyl diphosphate and dimethylallyl diphosphate precursors. The 13C-distributions also suggested cyclization of the 2,3-oxidosqualene precursor into the solanidine aglycone backbone involving a non–stereoselective hydroxylation step of the sterol a mixture of 25S-/25R-epimers of the SGAs.

Photosynthetic carbon allocation to live roots increases the year following high intensity defoliation across two ecosites in a temperate mixed grassland

Livestock grazing is an important driver of the carbon (C) cycle in grasslands and could be a decisive factor determining whether grasslands are a net sink or source of C. However, short-term C dynamics in grassland ecosystems and its response to different grazing intensities remain unclear, especially under field conditions. Here we test the hypothesis that photosynthetic C allocation in the plant-soil system of grasslands is differentially affected by the intensity of grazing that occurred in the prior growing season. We studied the fate of new C assimilates, using a 13CO2 pulse-labeling technique, as they move from shoots to roots and soil in vegetation previously exposed to different defoliation (emulated grazing) intensities in two semiarid Mixed Prairie grassland ecosites having contrasting soil textures (sandy vs. loamy). Within 24 h after photosynthetic uptake, 34.5% of the labeled C was translocated out of shoots, with 21% of the labeled C found in the mineral soil. Plant communities treated with high intensity defoliation during the previous growing season had more of the newly fixed 13C translocate into live roots at both ecosites throughout the 25-day chase period, indicating a higher demand for photosynthetic C by the root system of heavily defoliated plants. Twenty-five days after labeling, 13C transfer into the top 30 cm mineral soil was consistently greater at the loamy (26% of total 13C remaining) than at the sandy ecosite (10.4% of 13C remaining). We conclude that conditioning with high intensity defoliation the prior growing season stimulated short-term C allocation to roots in these semi-arid grasslands. Although this response was independent of soil texture, more C was translocated belowground overall in the loamy ecosite. The effect of defoliation intensity and ecosite in regulating short-term C allocation in the plant-soil system in temperate grasslands should be incorporated into future models describing C cycling in grasslands when predicting C dynamics under different management strategies employing contrasting grazing intensities.

Newly assimilated carbon allocation in grassland communities under different grazing enclosure times

The flow of photosynthesized carbon (C) from plants to soil and its loss from the respiration in the soil-plant system were measured via in situ 13CO2 pulse labeling pasture in overgrazed and long-term grazing exclosures (where grazing was not permitted for 5, 10, and 30 years). During a 30-day chase period, the allocation of newly assimilated C in the shoots, roots, and soil of grazing-excluded grasslands was higher than that in overgrazed grasslands. But the newly assimilated C allocated to shoots and soil respiration in overgrazed grasslands was significantly higher than that in grazing-excluded grasslands. Thirty days after labeling, 35% and 40% of the newly assimilated C in overgrazed grasslands were lost through shoots and soil respiration, respectively. The shoot 13C recovery was 32% in the 30 years 30 days after labeling, which was significantly higher than that obtained at the 5 and 10 years exclosure sites. Root and soil 13C recoveries in the 5- and 10-year exclosures were higher than those at the 30-year sites. In the grazing-excluded grasslands, the amount of 13C in soil organic C peaked 3 days after 13CO2 exposure and became relatively stable after 19 days of assimilation. The mean C residence time for non-structural C used in the soil respiration under overgrazing (10 days) was shorter than that under 5- (20 days) and 10- (17 days) or 30-year exclosures (14 days). Compared to overgrazing, grazing exclosure, especially moderate grazing exclosure, led to more allocation of newly assimilated C in the plant-soil system but fewer allocation in shoots and soil respiration.

Biosynthesis of salvinorin A

Salvia divinorum plants were exposed to a synthetic atmosphere containing 600 ppm 13CO2 for 4 hours with white light illumination and were subsequently cultured for a chase period of 10 days under standard greenhouse conditions. Salvinorin A was obtained by solvent extraction of the leaves and was purified chromatographically. The isotopolgue composition of salvinorin A was determined by NMR spectroscopy. The presence of multiply 13C-labeled isotopologues in considerable excess over their natural occurrence showed that the 13C labeling pulse had afforded multiply 13C-labeled biosynthate. Consecutive catabolic and anabolic reactions during the chase period afforded salvinorin A that was a mosaic revealing its origin from a mixture of labeled and unlabeled precursors. The presence of triple labeled isotopologues demonstrated that the IPP and DMAPP precursors of the diterpene, salvinorin A, were predominantly biosynthesized via the recently discovered non-mevalonate pathway.

Suppression of long chain acyl-CoA synthetase blocks intracellular fatty acid flux and glucose uptake in skeletal myotubes

Alterations in fatty acid metabolism are associated with impaired glucose uptake in skeletal muscle. Long-chain acyl-CoA synthetase (Acsl) 6 is the one of the Acsl isoforms expressed in skeletal muscle although its role in muscle energy metabolism has not been studied. Thus, the aims of this study were to investigate the role of Acsl6 in fatty acid partitioning and glucose uptake in differentiated skeletal myotubes using a siRNA-mediated knockdown approach. Compared with cells transfected with control siRNA, cells transfected with Acsl6 siRNA exhibited reduced intracellular triacylglycerol (TAG) accumulation. The initial rate of [1‑14C]‑oleic acid uptake was not altered while the incorporation of [1‑14C]‑acetic acids into total cellular lipids decreased under Acsl6 knockdown (p < 0.05). In a metabolic labeling study, Acsl6 suppression decreased the incorporation of [1‑14C]‑oleic acids and [1‑14C]‑acetic acids into TAG and diacylglycerol (DAG) (p < 0.05). During the chase period of a pulse-chase experiment, Acsl6 suppression increased the intracellular free fatty acids and decreased the fatty acid channeling toward the reacylation of TAG (p < 0.05). The incorporation of the labeled fatty acids into acid-soluble metabolites, β-oxidation product, was not changed under Acsl6 knockdown. Acsl6 siRNA decreased the insulin-induced uptake of [1‑14C]‑2‑deoxyglucose (p < 0.05) but did not change the glucose uptake in the presence of acipimox, inhibitor of lipolysis. Suppression of Acsl6 deteriorated Akt phosphorylation and Glut4 mRNA expression in response to insulin. These results suggest that Acsl6 activates and channels fatty acids toward anabolic pathways and has a role in glucose and fatty acid cycling through the re-esterification of fatty acids in skeletal muscle.

Proliferation and Differentiation of Gastric Mucous Neck and Chief Cells During Homeostasis and Injury-induced Metaplasia

Adult zymogen-producing (zymogenic) chief cells (ZCs) in the mammalian gastric gland base are believed to arise from descending mucous neck cells, which arise from stem cells. Gastric injury, such as from Helicobacter pylori infection in patients with chronic atrophic gastritis, can cause metaplasia, characterized by gastric cell expression of markers of wound-healing; these cells are called spasmolytic polypeptide-expressing metaplasia (SPEM) cells. We investigated differentiation and proliferation patterns of neck cells, ZCs, and SPEM cells in mice. C57BL/6 mice were given intraperitoneal injections of high-dose tamoxifen to induce SPEM or gavaged with H pylori (PMSS1) to induce chronic gastric injury. Mice were then given pulses of 5-bromo-2'-deoxyuridine (BrdU) in their drinking water, followed by chase periods without BrdU, or combined with intraperitoneal injections of 5-ethynyl-2'-deoxyuridine. We collected gastric tissues and performed immunofluorescence and immunohistochemical analyses to study gastric cell proliferation, differentiation, and turnover. After 8 weeks of continuous BrdU administration, fewer than 10% of homeostatic ZCs incorporated BrdU, whereas 88% of neck cells were labeled. In pulse-chase experiments, various chase periods decreased neck cell label but did not increase labeling of ZCs. When mice were given BrdU at the same time as tamoxifen, more than 90% of cells were labeled in all gastric lineages. After 3 months' recovery (no tamoxifen), ZCs became the predominant BrdU-labeled population, whereas other cells, including neck cells, were mostly negative. When we tracked the labeled cells in such mice over time, we observed that the proportion of BrdU-positive ZCs remained greater than 60% up to 11 months. In mice whose ZCs were the principal BrdU-positive population, acute injury by tamoxifen or chronic injury by H pylori infection resulted in SPEM cells becoming the principal BrdU-positive population. After withdrawal of tamoxifen, BrdU-positive ZCs reappeared. We studied mice in homeostasis or with tamoxifen- or H pylori-induced SPEM. Our findings indicated that mucous neck cells do not contribute substantially to generation of ZCs during homeostasis and that ZCs maintain their own census, likely through infrequent self-replication. After metaplasia-inducing injury, ZCs can become SPEM cells, and then redifferentiate into ZCs on injury resolution.

Fate of photosynthesized carbon as regulated by long–term tillage management in a dryland wheat cropping system

Tracking photosynthesized carbon (C) allocation into different C pools is crucial for management of C sequestration, especially in agroecosystems. However, the effects of long–term tillage management on allocation and dynamics of recently fixed C in the crop–soil–atmosphere system have been rarely investigated under dryland conditions. Using in–situ 13CO2 repeat labeling, this study quantified the photosynthesized C input to soil, and assessed the responses of allocation dynamics, microbial utilization, and aggregate protection of newly fixed C in three long–term (>10y) tillage practices (no–till, chisel–till, and plow–till) in a dryland wheat cropping system. Regardless of tillage practice, the 13C included into the shoots, roots, soil respiration, rhizosphere, and bulk soils accounted for 46–64%, 4.6–6.0%, 17–38%, 6.4–11.7%, and 2.8–7.2%, respectively, of added 13C over a 35–d chase period. Owing to relatively low plant biomass, compared to plow–till, long–term no–till and chisel–till on average lowered plant 13C fixation and its allocation in rhizosphere soil by 17% and 11%, and 21% and 15%, respectively. Nevertheless, the 13C relocated to bulk soil was significantly higher under both no–till (0.24 g m−2) and chisel–till (0.22 g m−2) than that under plow–till (0.18 g m−2) over the chase period. This could be partially attributed to the decreased allocation of belowground 13C to root–derived CO2 releases and the increased C retention in both macroaggregates and microaggregates in conservation tillage plots. Moreover, the reduced microbial utilization of new C in rhizosphere soil, as indicated by the low 13C amount in microbial biomass, may further facilitate new C accumulation in bulk soil under reduced tillage. Our findings suggest that despite low photosynthetically–fixed C input to belowground pools during the early growth stages, soils under long–term conservation tillage, in particular no–till, can have a distinct efficiency advantage in soil carbon sequestration by enhancing photosynthesized C preservation in soil aggregates and decreasing new C loss from root–derived CO2 release in the dryland wheat–soil system.

Effects of nitrogen fertilization on pot‐grown wheat photosynthate partitioning within intensively farmed soil determined by 13C pulse‐labeling

AbstractUnderstanding rhizodeposited carbon (C) dynamics of winter wheat (Triticum aestivum L.) is important for improving soil fertility and increasing soil C stocks. However, the effects of nitrogen (N) fertilization on photosynthate C allocation to rhizodeposition of wheat grown in an intensively farmed alkaline soil remain elusive. In this study, pot‐grown winter wheat under N fertilization of 250 kg N ha−1 was pulse‐labeled with 13CO2 at tillering, elongation, anthesis, and grain‐filling stages. The 13C in shoots, roots, soil organic carbon (SOC), and rhizosphere‐respired CO2 was measured 28 d after each 13C labeling. The proportion of net‐photosynthesized 13C recovered (shoots + roots + soil + soil respired CO2) in the shoots increased from 58–64% at the tillering to 86–91% at the grain‐filling stage. Likewise, the proportion in the roots decreased from 21–28% to 2–3%, and that in the SOC pool increased from 1–2% to 6–7%. However, the 13C respired CO2 allocated to soil peaked (17–18%) at the elongation stage and decreased to 6–8% at the grain‐filling stage. Over the entire growth season of wheat, N fertilization decreased the proportion of net photosynthate C translocated to the below‐ground pool by about 20%, but increased the total amount of fixed photosynthate C, and therefore increased the below‐ground photosynthate C input. We found that the chase period of about 4 weeks is sufficient to accurately monitor the recovery of 13C after pulse labeling in a wheat–soil system. We conclude that N fertilization increased the deposition of photoassimilate C into SOC pools over the entire growth season of wheat compared to the control treatment.

P-SELECTIN EXPRESSION MARKS ACTIVE AND MYELOID-BIASED HEMATOPOIETIC STEM CELLS AND IS UP-REGULATED WITH AGE

Hematopoietic stem cells (HSCs) contribute to blood formation throughout the entire lifespan of an organism, while simultaneously sustaining the stem cell pool. Aged HSCs increase in number while the functional activity of aged HSCs associated with a skewed differentiation potential decreases substantially compared to their young counterparts. Multiple studies have reported that P-selectin (Selp) is consistently upregulated in murine aged HSCs. However, the role of Selp in HSCs aging is not clear. Selp preferentially marks CD150high Lin-Sca-1+ckit+CD48- HSCs, which are myeloid-biased. Beyond an increase of the expression levels of Selp by aged HSCs, the number of Selp+ HSCs expands strongly with age. We in vivo pulsed HSCs using the TetOP-H2B-GFP mouse model in which quiescent HSCs retain the GFP label after an extended chase period. We found that label-retaining GFP+ HSCs show low Selp expression, while more active GFP- HSCs are Selp+. To further investigate functional differences between Selp- and Selp+ HSCs, we separated these two populations from aged donor mice, and transplanted them into lethally irradiated recipients. Preliminary data show that Selp+ HSCs are more myeloid-biased, which is in line with high Selp expression on CD150high HSCs. Therefore, we suggest that Selp- HSCs isolated from an aged donor are functionally 'younger' stem cells. We are currently performing RNA-seq on Selp- and Selp+ aged HSCs to provide further molecular insight into the functional differences between these two highly related, yet differentially aged HSC subsets.

Allocation of assimilated carbon in paddies depending on rice age, chase period and N fertilization: Experiment with 13CO2 labelling and literature synthesis

Quantification of total belowground carbon (C) input and allocation to various pools in rice–soil systems depending on plant age, chase period, and nitrogen (N) availability. Rice plants with and without N fertilization were 13CO2 pulse labelled at tillering, elongation, heading, and filling stages and were destructively sampled after 6 h of labelling and at the final harvest. The allocation of C by rice was also generalized based on literature pertaining to 94 studies with respect to plant age and chase period. The C allocation in roots and soil strongly decreased with plant age. The literature review showed that C allocation to roots increased within the first 10 days of labelling and remained stable thereafter. Nitrogen fertilization had no effect on C allocation immediately after assimilation, but increased C remained belowground at 1.7-times that at final harvest. The total belowground net C input by one rice crop was 630–1080 kg C ha−1, including rhizodeposition of 160–330 kg C ha−1. Multiple pulse labelling at various plant growth stages and taking multiple subsequent samples as well as nutrient availability should be considered for tracing C flows more accurately for precise C balance in rice paddy systems.

Tamoxifen Improves Glucose Tolerance in a Delivery-, Sex-, and Strain-Dependent Manner in Mice.

Tamoxifen, a selective estrogen-receptor modulator, is widely used in mouse models to temporally control gene expression but is also known to affect body composition. We report that tamoxifen has significant and sustained effects on glucose tolerance, independent of effects on insulin sensitivity, in mice. IP, but not oral, tamoxifen delivery improved glucose tolerance in three inbred mouse strains. The extent and persistence of tamoxifen-induced effects were sex and strain dependent. These findings highlight the need to revise commonly used tamoxifen-based protocols for gene manipulation in mice by including longer chase periods after injection, oral delivery, and the use of tamoxifen-treated littermate controls.

Plant-microbe competition: does injection of isotopes of C and N into the rhizosphere effectively characterise plant use of soil N?

Despite considerable attention over the last 25yr, the importance of early protein breakdown products to plant nitrogen (N) nutrition remains uncertain. We used rhizosphere injection of 15 N-, 13 C- and 14 C-labelled inorganic N and amino acid (l-alanine), with chase periods from 1min to 24h, to investigate the duration of competition for amino acid between roots (Triticum aestivum) and soil microorganisms. We further investigated how microbial modification of l-alanine influenced plant carbon (C) and N recovery. From recovery of C isotopes, intact alanine uptake was 0.2-1.3% of added. Soil microbes appeared to remove alanine from soil solution within 1min and release enough NH4+ to account for all plant 15 N recovery (over 24h) within 5min. Microbially generated inorganic or keto acid C accounted for <25% of the lowest estimate of intact alanine uptake. Co-location of C and N labels appears a reasonable measure of intact uptake. Potential interference from microbially modified C is probably modest, but may increase with chase period. Similarly, competition for l-alanine is complete within a few minutes in soil, whereas NO3- added at the same rate is available for >24h, indicating that long chase periods bias outcomes and fail to accurately simulate soil processes.

Monitoring of the Cytoskeleton-Dependent Intracellular Trafficking of Fluorescent Iron Oxide Nanoparticles by Nanoparticle Pulse-Chase Experiments in C6 Glioma Cells.

Iron oxide nanoparticles (IONPs) are used for various biomedical and therapeutic approaches. To investigate the uptake and the intracellular trafficking of IONPs in neural cells we have performed nanoparticle pulse-chase experiments to visualize the internalization and the fate of fluorescent IONPs in C6 glioma cells and astrocyte cultures. Already a short exposure to IONPs for 10min at 4°C (nanoparticle pulse) allowed binding of substantial amounts of nanoparticles to the cells, while internalization of IONPs into the cell was prevented. The uptake of bound IONPs and the intracellular trafficking was started by increasing the temperature to 37°C (chase period). While hardly any cellular fluorescence nor any iron staining was detectable directly after the nanoparticle pulse, dotted cellular fluorescence and iron patterns appeared already within a few minutes after start of the chase incubation and became intensified in the perinuclear region during further incubation for up to 90min. Longer chase incubations resulted in separation of the fluorescent coat from the core of the internalized IONPs. Disruption of actin filaments in C6 cells strongly impaired the internalization of IONPs, whereas destabilization of microtubules traped IONP-containing vesicles to the plasma membrane. In conclusion, nanoparticle pulse-chase experiments allowed to synchronize the cellular uptake of fluorescent IONPs and to identify for C6 cells an actin-dependent early and a microtubule-dependent later process in the intracellular trafficking of fluorescent IONPs.

Longevity of Beta Cells and Protein Complexes Revealed by Isotope Imaging

We discovered recently that long-lived cells (LLCs), such as neurons, contain long-lived proteins (LLPs) in the nucleus that can last a lifetime, and their decline during aging impairs nuclear function. Beta cells are LLCs with a slow cell replication process, yet it is unknown whether individual beta cells have different lifespans. We tested this hypothesis by using a hybrid microscopy and mapping platform (MIMS-EM) to identify beta cells and quantify cell and protein turnover in the islets of Langerhans. This is achieved by pulse-labelling mice with an 15N-rich diet followed by a chase period with a control 14N-rich diet for up to 2 years. Here, old cells and protein structures retain 15N while those that turnover incorporate 14N. The levels of 15N and 14N were measured with multi-isotope mass spectroscopy (MIMS) and the 15N-to-14N ratio reports on cell and protein turnover. Importantly, the MIMS data is correlated with scanning electron microscopy (EM) to create high-resolution maps of cells and intra-cellular structures overlaid with age information. Here we report the use of MIMS-EM on mouse islets to discover that beta cells have vastly different ages. In fact, while approximately 75% of beta cells replicate between 2-4 times, almost 25% of all beta cells does not replicate at all after weaning and remain quiescent for an entire lifetime, and therefore, are as old as cortical neurons. Such longevity was also found in other islet cell-types and at the protein super-complex level, where 15N accumulation on the primary cilium of beta cells strongly indicates that this important signaling structure is exceptionally long-lived. Strikingly, and similar to what is found in old rat neurons, beta cells from humans aged &amp;gt;60-years-old lose specific nuclear LLPs and show signs of impaired nuclear function. Together, our data shows that beta cell longevity is heterogeneous and identifies the loss of nuclear LLPs in human beta cells as a potential contributing factor to age-dependent loss of beta cell function. Disclosure R. Arrojo e Drigo: Stock/Shareholder; Self; VTV Therapeutics, Novo Nordisk A/S. B.H. Toyama: None. S. Tyagi: None. V. Lev-Ram: None. T.J.J. Deerinck: None. E.A. Bushong: None. R. Ramachandra: None. C. Lechene: None. M.H. Ellisman: None. M. Hetzer: None.