Elevated CO2 promotes long‐term nitrogen accumulation only in combination with nitrogen addition

Biogeochemical models that incorporate nitrogen (N) limitation indicate that N availability will control the magnitude of ecosystem carbon uptake in response to rising CO₂. Some models, however, suggest that elevated CO₂ may promote ecosystem N accumulation, a feedback that in the long term could circumvent N limitation of the CO₂ response while mitigating N pollution. We tested this prediction using a nine‐year CO₂xN experiment in a tidal marsh. Although the effects of CO₂ are similar between uplands and wetlands in many respects, this experiment offers a greater likelihood of detecting CO₂ effects on N retention on a decadal timescale because tidal marshes have a relatively open N cycle and can accrue soil organic matter rapidly. To determine how elevated CO₂ affects N dynamics, we assessed the three primary fates of N in a tidal marsh: (1) retention in plants and soil, (2) denitrification to the atmosphere, and (3) tidal export. We assessed changes in N pools and tracked the fate of a ¹⁵N tracer added to each plot in 2006 to quantify the fraction of added N retained in vegetation and soil, and to estimate lateral N movement. Elevated CO₂ alone did not increase plant N mass, soil N mass, or ¹⁵N label retention. Unexpectedly, CO₂ and N interacted such that the combined N+CO₂ treatment increased ecosystem N accumulation despite the stimulation in N losses indicated by reduced ¹⁵N label retention. These findings suggest that in N‐limited ecosystems, elevated CO₂ is unlikely to increase long‐term N accumulation and circumvent progressive N limitation without additional N inputs, which may relieve plant–microbe competition and allow for increased plant N uptake.     

Hydraulic lift of Medicago sativa and Astragalus laxmannii and its effect on their neighborhood plants

为探讨紫花苜蓿(Medicago sativa)及斜茎黄耆(沙打旺, Astragalus laxmannii)与禾本科牧草混播后的水力提升现象, 揭示深、浅根性牧草的种间关系, 为混播草地的建植提供理论依据, 该研究开展了室外“上下盆”分根盆栽试验、采用土壤水分测定及“重水” (D₂O, 氘(D)含量>99.9%)标记法估算了苜蓿及斜茎黄耆分别与‘冬牧70’黑麦(Secale cereal ‘Dongmu 70’)不同混播比例(豆科:禾本科分别为3:7、5:5、7:3)条件下水力提升的发生情况及其对伴生牧草生长生理性状的影响。结果表明: ‘冬牧70’黑麦与斜茎黄耆混播后的产量显著高于其与紫花苜蓿混播后的产量, 同一种禾豆牧草混播组合不同混播比例中, 以AC2 (紫花苜蓿:‘冬牧70’黑麦为5:5)和BC3 (斜茎黄耆:‘冬牧70’黑麦为7:3)混播组合的总产量最高。不同单混播组合的单株整个生育期内日均提水量存在显著差异, 两种豆科牧草在混播时日均提水量均高于单播时, 斜茎黄耆单混播时的日均提水量显著高于紫花苜蓿, BC2组合(斜茎黄耆:‘冬牧70’黑麦为5:5)的日均提水量高于其他混播组合。在用标记水处理下盆土壤后, 各组合上下盆土壤水氢稳定同位素比率(δD)值显著升高。不同禾豆牧草组合上盆土壤水δD及禾本科牧草茎秆水δD、整株碳同位素分辨率(Δ¹³C)和产量数据表明, 在斜茎黄耆与‘冬牧70’黑麦混播比例为3:7、紫花苜蓿与‘冬牧70’黑麦混播比例为5:5时, 禾本科牧草水分状况或产量好于其他混播比例。以上结果表明, 两种深浅根豆科牧草与浅根性禾本科牧草混播种植时发生了水力提升现象, 两种豆科牧草提升的水分可以被伴生的禾本科牧草所吸收利用。     

The flux ratio equation under nonstationary conditions

Summary The time dependent (i.e., nonstationary) unidirectional fluxes through a multilayered system consisting of sandwiched layers of arbitrary composition and exhibiting arbitrary potential and resistance profiles have been calculated, assuming that the flux is governed by the Smoluchowski equation (i.e., a flux resulting from a diffusion process superimposed upon a migration and/or a convection process, where part of the latter may arise from an active transport process). It is shown that during the building up of the concentration profile of the isotope inside the system towards the stationary value the ratio between the two oppositely directed, time-dependent unidirectional fluxes is, from the very first appearance of the isotope in the surrounding solutions, equal to the value of the stationary flux ratio. The practical implications of this result are discussed.     

Lack of photosynthetic or stomatal regulation after 9 years of elevated [CO2] and 4 years of soil warming in two conifer species at the alpine treeline

Alpine treelines are temperature‐limited vegetation boundaries. Understanding the effects of elevated [CO₂] and warming on CO₂ and H₂O gas exchange may help predict responses of treelines to global change. We measured needle gas exchange of Larix decidua Mill. and Pinus mugo ssp. uncinata DC trees after 9 years of free air CO₂ enrichment (575 µmol mol^?1) and 4 years of soil warming (+4 °C) and analysed δ¹³C and δ¹⁸O values of needles and tree rings. Tree needles under elevated [CO₂] showed neither nitrogen limitation nor end‐product inhibition, and no down‐regulation of maximal photosynthetic rate (A_max) was found. Both tree species showed increased net photosynthetic rates (A_n) under elevated [CO₂] (L. decidua: +39%; P. mugo: +35%). Stomatal conductance (g_H2O) was insensitive to changes in [CO₂], thus transpiration rates remained unchanged and intrinsic water‐use efficiency (iWUE) increased due to higher A_n. Soil warming affected neither A_n nor g_H2O. Unresponsiveness of g_H2O to [CO₂] and warming was confirmed by δ¹⁸O needle and tree ring values. Consequently, under sufficient water supply, elevated [CO₂] induced sustained enhancement in A_n and lead to increased C inputs into this ecosystem, while soil warming hardly affected gas exchange of L. decidua and P. mugo at the alpine treeline.     

Synthesis and evaluation of (−)- and (+)-[11C]galanthamine as PET tracers for cerebral acetylcholinesterase imaging

Improved radiopharmaceuticals for imaging cerebral acetylcholinesterase (AChE) are needed for the diagnosis of Alzheimer’s disease (AD). Thus, ¹¹C-labeled (−)-galanthamine and its enantiomers were synthesized as novel agents for imaging the localization and activity of AChE by positron emission tomography (PET). C-11 was incorporated into (−)- and (+)-[¹¹C]galanthamine by N-methylation of norgalanthamines with [¹¹C]methyl triflate. Simple accumulation of ¹¹C in the brain was measured in an in vivo biodistribution study using mice, whilst donepezil was used as a blocking agent in analogous in vivo blocking studies. In vitro autoradiography of rat brain tissue was performed to investigate the distribution of (−)-[¹¹C]galanthamine, and confirmed the results of PET studies in mice. The radiochemical yields of N-methylation of (−)- and (+)-norgalanthamines were 13.7% and 14.4%, respectively. The highest level of accumulation of ¹¹C in the brains of mice was observed at 10 min after administration (2.1% ID/g). Intravenous pretreatment with donepezil resulted in a 30% decrease in accumulation of (−)-[¹¹C]galanthamine in the striatum; however, levels in the cerebellum were unchanged. In contrast, use of (+)-[¹¹C]galanthamine led to accumulation of radioactivity in the striatum equal to that in the cerebellum, and these levels were unaffected by pretreatment with donepezil. In in vitro autoradiography of regional radioactive signals of brain sections showed that pretreatment with either (−)-galanthamine or donepezil blocked the binding of (−)-[¹¹C]galanthamine to the striatum, while sagittal PET imaging revealed accumulation of (−)-[¹¹C]galanthamine in the brain. These results indicate that (−)-[¹¹C]galanthamine showed specific binding to AChE, whereas (+)-[¹¹C]-galanthamine accumulated in brain tissue by non-specific binding. Thus, optically pure (−)-[¹¹C]galanthamine could be a useful PET tracer for imaging cerebral AChE.     

Synthesis and evaluation of 18F-labeled mitiglinide derivatives as positron emission tomography tracers for β-cell imaging

Measuring changes in β-cell mass in vivo during progression of diabetes mellitus is important for understanding the pathogenesis, facilitating early diagnosis, and developing novel therapeutics for this disease. However, a non-invasive method has not been developed. A novel series of mitiglinide derivatives (o-FMIT, m-FMIT and p-FMIT; FMITs) were synthesized and their binding affinity for the sulfonylurea receptor 1 (SUR1) of pancreatic islets were evaluated by inhibition studies. (+)-(S)-o-FMIT had the highest affinity of our synthesized FMITs (IC₅₀ = 1.8 μM). (+)-(S)-o-[¹⁸F]FMIT was obtained with radiochemical yield of 18% by radiofluorination of racemic precursor 7, hydrolysis, and optical resolution with chiral HPLC; its radiochemical purity was >99%. In biodistribution experiments using normal mice, (+)-(S)-o-[¹⁸F]FMIT showed 1.94 ± 0.42% ID/g of pancreatic uptake at 5 min p.i., and decreases in radioactivity in the liver (located close to the pancreas) was relatively rapid. Ex vivo autoradiography experiments using pancreatic sections confirmed accumulation of (+)-(S)-o-[¹⁸F]FMIT in pancreatic β-cells. These results suggest that (+)-(S)-o-[¹⁸F]FMIT meets the basic requirements for an radiotracer, and could be a candidate positron emission tomography tracer for in vivo imaging of pancreatic β-cells.     

Emerging Technologies to Map the Protein Methylome

Protein methylation plays an integral role in cellular signaling, most notably by modulating proteins bound at chromatin and increasingly through regulation of non-histone proteins. One central challenge in understanding how methylation acts in signaling is identifying and measuring protein methylation. This includes locus-specific modification of histones, on individual non-histone proteins, and globally across the proteome. Protein methylation has been studied traditionally using candidate approaches such as methylation-specific antibodies, mapping of post-translational modifications by mass spectrometry, and radioactive labeling to characterize methylation on target proteins. Recent developments have provided new approaches to identify methylated proteins, measure methylation levels, identify substrates of methyltransferase enzymes, and match methylated proteins to methyl-specific reader domains. Methyl-binding protein domains and improved antibodies with broad specificity for methylated proteins are being used to characterize the “protein methylome”. They also have the potential to be used in high-throughput assays for inhibitor screens and drug development. These tools are often coupled to improvements in mass spectrometry to quickly identify methylated residues, as well as to protein microarrays, where they can be used to screen for methylated proteins. Finally, new chemical biology strategies are being used to probe the function of methyltransferases, demethylases, and methyl-binding “reader” domains. These tools create a “system-level” understanding of protein methylation and integrate protein methylation into broader signaling processes.     

In vitro production of radiolabeled red clover (<Emphasis Type="Italic">Trifolium pratense</Emphasis>) isoflavones

Red clover isoflavones are increasingly used in dietary supplements for their purported estrogenic effects. However, little is known about their metabolism in animals due to a lack of commercially available isotopically labeled tracers. The goal of this research was to establish red clover cell culturing methodology for ¹⁴C-biolabeling of isoflavones. When root, leaf, and petiole-derived suspension cultures were grown in darkness or light, dark-grown, petiole-derived solution cultures produced the highest concentrations of the two major red clover isoflavones, formononetin (0.67 mg/g FM inoculum) and biochanin A (0.13 mg/g FM inoculum). Varying levels and timing of copper chloride elicitor did not significantly affect isoflavone accumulation. Approximately 38% of the ¹⁴C-sucrose dose accumulated in the cells. Eighteen percent of the initial labeled dose was detected in the isoflavone-rich methanolic extract and of that, 22% accumulated in isoflavones.     

Leaf carbon management in slow-growing plants exposed to elevated CO2

Two slow‐growing plant species (Chamaerops humilis, L. and Cycas revoluta Thunb.) were exposed to elevated CO₂ conditions over a 20‐month period in order to study the CO₂ effect on growth, photosynthetic capacity and leaf carbon (C) management. The ambient isotopic ¹³C/¹²C composition (δ¹³C) of the greenhouse module corresponding to elevated CO₂ (800 μmol mol^?1 CO₂) conditions was changed from δ¹³C ca. ?12.8±0.3‰ to ca. ?19.2±0.2‰. Exposure of these plants to elevated CO₂ enhanced dry mass (DM) by 82% and 152% in Chamerops and Cycas, respectively, mainly as a consequence of increases in plant level photosynthetic rates. However, analyses of A–C_i curve parameters revealed that elevated CO₂ diminished leaf photosynthetic rates of Chamaerops whereas in Cycas, no photosynthetic acclimation was detected. The fact that Chamaerops plants had a lower DM increase, together with a longer leaf C residence time and a diminished capacity to respire recently fixed C, suggests that this species was unable to increase C sink strength. Furthermore, the consequent C source/sink imbalance in Chamaerops might have induced the downregulation of Rubisco. Cycas plants were capable of avoiding photosynthetic downregulation due to a greater ability to increase C sink strength, as was confirmed by DM values, and ¹²C‐enriched CO₂ labeling data. Cycas developed the ability to respire a larger proportion of recently fixed C and to reallocate the recently fixed C away from leaves to other plant tissues. These findings suggest that leaf C management is a key factor in the responsiveness of slow‐growing plants to future CO₂ scenarios.     

Role of specific residues in coenzyme binding, charge-transfer complex formation, and catalysis in Anabaena ferredoxin NADP-reductase

Two transient charge-transfer complexes (CTC) form prior and upon hydride transfer (HT) in the reversible reaction of the FAD-dependent ferredoxin-NADP⁺ reductase (FNR) with NADP⁺/H, FNR_ox-NADPH (CTC-1), and FNR_rd-NADP⁺ (CTC-2). Spectral properties of both CTCs, as well as the corresponding interconversion HT rates, are here reported for several Anabaena FNR site-directed mutants. The need for an adequate initial interaction between the 2′P-AMP portion of NADP⁺/H and FNR that provides subsequent conformational changes leading to CTC formation is further confirmed. Stronger interactions between the isoalloxazine and nicotinamide rings might relate with faster HT processes, but exceptions are found upon distortion of the active centre. Thus, within the analyzed FNR variants, there is no strict correlation between the stability of the transient CTCs formation and the rate of the subsequent HT. Kinetic isotope effects suggest that, while in the WT, vibrational enhanced modulation of the active site contributes to the tunnel probability of HT; complexes of some of the active site mutants with the coenzyme hardly allow the relative movement of isoalloxazine and nicotinamide rings along the HT reaction. The architecture of the WT FNR active site precisely contributes to reduce the stacking probability between the isoalloxazine and nicotinamide rings in the catalytically competent complex, modulating the angle and distance between the N5 of the FAD isoalloxazine and the C4 of the coenzyme nicotinamide to values that ensure efficient HT processes.