Elemental formula annotation of polar and lipophilic metabolites using (13) C, (15) N and (34) S isotope labelling, in combination with high-resolution mass spectrometry

The unbiased and comprehensive analysis of metabolites in any organism presents a major challenge if proper peak annotation and unambiguous assignment of the biological origin of the peaks are required. Here we provide a comprehensive multi-isotope labelling-based strategy using fully labelled (13) C, (15) N and (34) S plant tissues, in combination with a fractionated metabolite extraction protocol. The extraction procedure allows for the simultaneous extraction of polar, semi-polar and hydrophobic metabolites, as well as for the extraction of proteins and starch. After labelling and extraction, the metabolites and lipids were analysed using a high-resolution mass spectrometer providing accurate MS and all-ion fragmentation data, providing an unambiguous readout for every detectable isotope-labelled peak. The isotope labelling assisted peak annotation process employed can be applied in either an automated database-dependent or a database-independent analysis of the plant polar metabolome and lipidome. As a proof of concept, the developed methods and technologies were applied and validated using Arabidopsis thaliana leaf and root extracts. Along with a large repository of assigned elemental compositions, which is provided, we show, using selected examples, the accuracy and reliability of the developed workflow.     

Construction of an E. Coli genome-scale atom mapping model for MFA calculations

Metabolic flux analysis (MFA) has so far been restricted to lumped networks lacking many important pathways, partly due to the difficulty in automatically generating isotope mapping matrices for genome-scale metabolic networks. Here we introduce a procedure that uses a compound matching algorithm based on the graph theoretical concept of pattern recognition along with relevant reaction information to automatically generate genome-scale atom mappings which trace the path of atoms from reactants to products for every reaction. The procedure is applied to the iAF1260 metabolic reconstruction of Escherichia coli yielding the genome-scale isotope mapping model imPR90068. This model maps 90,068 non-hydrogen atoms that span all 2,077 reactions present in iAF1260 (previous largest mapping model included 238 reactions). The expanded scope of the isotope mapping model allows the complete tracking of labeled atoms through pathways such as cofactor and prosthetic group biosynthesis and histidine metabolism. An EMU representation of imPR90068 is also constructed and made available.     

能量标签技术及其在红树林生态系统能流研究中的应用

传统上认为红树林输出的有机质产生巨大的能流,支持了巨大的河口和近岸水域生态系统的次级生产。但能量标签技术的研究结果却显示红树林输出的有机质的作用并没有如此巨大。用红树碎屑难消化特性来解释此现象,此外数学模型模拟分析发现潮汐的稀释作用也可以解释这种现象。但这两者都不能解释,在其他初级生产者稀少时,红树材输出的有机质可以被大量利用的现象。在有红树林的河口和近海岸水域生态系统中,藻类等非红树初级生产者具有比红树植物更高的初级生产力,而且更容易被动物获得和消化。可以认为是藻类等巨大初级生产力的竞争作用导致红树初级生产在消费者组织中很难被发现,如此上面提到的难题就能得到很好的解决。此外能量标签技术检测出的是红树的初级生产在消费者组织中的相对比率,不是绝对数量值,从此角度看,能量标签技术的结果与传统观点不是矛盾而是互相补充的关系。由此推测红树的初级生产应该还是被消费者所利用,只是它们在消费者初级营养来源组成中占的比例并不大,但其绝对数量并不少。这与传统观点认为的红树的初级生产被大量利用,支撑了具有巨大的次级生产稍有不同。此外,能量标签技术在红树林生态系统中的适用性尚未检验;计算食物组成的数学工具不是很完善;实验设计上考虑的不够全面;对定量研究有一定的影响。     

The isotope hydrology of Quaternary climate change

Understanding the links between climate change and human migration and culture is an important theme in Quaternary archaeology. While oxygen and hydrogen stable isotopes in high-latitude ice cores provide the ultimate detailed record of palaeoclimate extending back to the Middle Pleistocene, groundwater can act as a climate archive for areas at lower latitudes, permitting a degree of calibration for proxy records such as lake sediments, bones, and organic matter. Not only can oxygen and hydrogen stable isotopes be measured on waters, but the temperature of recharge can be calculated from the amount of the atmospheric noble gases neon, argon, krypton, and xenon in solution, while residence time can be estimated from the decay of the radioisotopes carbon-14, chlorine-36, and krypton-81 over timescales comparable to the ice core record. The Pleistocene-Holocene transition is well characterised in aquifers worldwide, and it is apparent that isotope-temperature relationships of the present day are not necessarily transferable to past climatic regimes, with important implications for the interpretation of proxy isotope data. Groundwaters dating back to one million years, i.e., to beyond the Middle Pleistocene, are only found in major aquifer basins and information is relatively sparse and of low resolution. Speleothem fluid inclusions offer a way of considerably increasing this resolution, but both speleothem formation and large-scale groundwater recharge requires humid conditions, which may be relatively infrequent for areas currently experiencing arid climates. Both types of record therefore require caution in their interpretation when considering a particular archaeological context.     

Climate controls on C3 vs. C4 productivity in North American grasslands from carbon isotope composition of soil organic matter

We analyzed the δ¹³C of soil organic matter (SOM) and fine roots from 55 native grassland sites widely distributed across the US and Canadian Great Plains to examine the relative production of C₃ vs. C₄ plants (hereafter %C₄) at the continental scale. Our climate vs. %C₄ results agreed well with North American field studies on %C₄, but showed bias with respect to %C₄ from a US vegetation database (statsgo ) and weak agreement with a physiologically based prediction that depends on crossover temperature. Although monthly average temperatures have been used in many studies to predict %C₄, our analysis shows that high temperatures are better predictors of %C₄. In particular, we found that July climate (average of daily high temperature and month's total rainfall) predicted %C₄ better than other months, seasons or annual averages, suggesting that the outcome of competition between C₃ and C₄ plants in North American grasslands was particularly sensitive to climate during this narrow window of time. Root δ¹³C increased about 1‰ between the A and B horizon, suggesting that C₄ roots become relatively more common than C₃ roots with depth. These differences in depth distribution likely contribute to the isotopic enrichment with depth in SOM where both C₃ and C₄ grasses are present.     

稳定性同位素探测技术在微生物生态学研究中的应用

稳定性同位素标记技术同分子生物学技术相结合而发展起来的稳定性同位素探测技术（stableisotope probing,SIP）,在对各种环境中微生物群落组成进行遗传分类学鉴定的同时,可确定其在环境过程中的功能,提供复杂群落中微生物相互作用及其代谢功能的大量信息,具有广阔的应用前景.其基本原理是：将原位或微宇宙（microcosm）的环境样品暴露于稳定性同位素富集的基质中,这些样品中存在的某些微生物能够以基质中的稳定（性同位素为碳源或氮源进行物质代谢并满足其自身生长需要,基质中的稳定性同位素被吸收同化进入微生物体内,参与各类物质如核酸（DNA和RNA）及磷脂脂肪酸（PLFA）等的生物合成,通过提取、分离、纯化、分析这些微生物体内稳定性同位素标记的生物标志物,从而将微生物的组成与其功能联系起来.在介绍稳定性同位素培养基质的选择及标记方法、合适的生物标志物的选择及提取分离方法的基础上,举例阐述了此项技术在甲基营养菌、有机污染物降解菌、根际微生物生态、互营微生物、宏基因组学等方面的应用.     

Plant functional types broadly describe water use strategies in the Caatinga,a seasonally dry tropical forest in northeast Brazil

1. In seasonally dry tropical forests, plant functional type can be classified as deciduous low wood density, deciduous high wood density, or evergreen high wood density species. While deciduousness is often associated with drought‐avoidance and low wood density is often associated with tissue water storage, the degree to which these functional types may correspond to diverging and unique water use strategies has not been extensively tested.
2. We examined (a) tolerance to water stress, measured by predawn and mid‐day leaf water potential; (b) water use efficiency, measured via foliar δ¹³C; and (c) access to soil water, measured via stem water δ¹⁸O.
3. We found that deciduous low wood density species maintain high leaf water potential and low water use efficiency. Deciduous high wood density species have lower leaf water potential and variable water use efficiency. Both groups rely on shallow soil water. Evergreen high wood density species have low leaf water potential, higher water use efficiency, and access alternative water sources. These findings indicate that deciduous low wood density species are drought avoiders, with a specialized strategy for storing root and stem water. Deciduous high wood density species are moderately drought tolerant, and evergreen high wood density species are the most drought tolerant group.
4. Synthesis. Our results broadly support the plant functional type framework as a way to understand water use strategies, but also highlight species‐level differences.

     

Isotopic fractionation with morphological change and sexual specificity in the lappet moth Euthrix potatoria

The δ¹⁵N values of adult holometabolous insects exceed those of larvae, but otherwise little information on terrestrial invertebrates has been obtained in food‐web analyses using stable isotope ratios (δ¹⁵N, δ¹³C). Changes in δ¹³C during metamorphosis and differences between males and females have not been examined. We collected the larvae and cocoons of Euthrix potatoria (L.) (Lepidoptera: Lasiocampidae) in the field and used them to assess the species’ isotopic fractionation. Each emerged moth was divided into five body parts. We conducted stable N and C isotope analyses for each body part, as well as for cocoons and exuviae, and also compared stable isotope ratios between sexes. We confirmed δ¹⁵N enrichment through metamorphosis and estimated that δ¹⁵N enrichment is accomplished by the relative concentration of ¹⁵N due to the excretion of copious meconium, which contains abundant ¹⁴N. We also observed changes in δ¹³C values through metamorphosis. Both isotope values tended to change more in males than in females. The proportion of the whole‐adult weight represented by meconium was higher in males than in females, suggesting that high meconium secretion in males contributes to the sexual difference in δ¹⁵N. These phenomena may be common in Holometabola, which require a pupal stage. For more accurate food‐web assessments, it is important to consider stable isotope changes during different life cycles, as well as sexual differences.     

Carbon partitioning in Arabidopsis thaliana is a dynamic process controlled by the plants metabolic status and its circadian clock

Plant growth involves the coordinated distribution of carbon resources both towards structural components and towards storage compounds that assure a steady carbon supply over the complete diurnal cycle. We used ¹⁴CO₂ labelling to track assimilated carbon in both source and sink tissues. Source tissues exhibit large variations in carbon allocation throughout the light period. The most prominent change was detected in partitioning towards starch, being low in the morning and more than double later in the day. Export into sink tissues showed reciprocal changes. Fewer and smaller changes in carbon allocation occurred in sink tissues where, in most respects, carbon was partitioned similarly, whether the sink leaf assimilated it through photosynthesis or imported it from source leaves. Mutants deficient in the production or remobilization of leaf starch exhibited major alterations in carbon allocation. Low‐starch mutants that suffer from carbon starvation at night allocated much more carbon into neutral sugars and had higher rates of export than the wild type, partly because of the reduced allocation into starch, but also because of reduced allocation into structural components. Moreover, mutants deficient in the plant's circadian system showed considerable changes in their carbon partitioning pattern suggesting control by the circadian clock.     

Association of nitrogen-fixing,plant-growth-promoting rhizobacteria (PGPR) with kallar grass and rice

Leptochloa fusca (L.) Kunth (kallar grass) has previously been found to exhibit high rates of nitrogen fixation. A series of experiments to determine the level of biological nitrogen fixation using N isotopic dilution were carried out in nutrient solution and saline soil. These studies indicated an agronomically significant amount of nitrogen being fixed in soil. Kallar grass has a similar growth habitat to rice. Therefore similar studies were carried out with rice after isolating various diazotrophs from the roots which were also screened for their ability to produce auxin (IAA). Five such strains namely Azospirillum lipoferum N-4, Azospirillum brasilense Wb-3, Azoarcus K-1, Pseudomonas 96-51, Zoogloea Ky-1 were selected for inoculating two rice varieties i.e. NIAB-6 and BAS-370 under aseptic laboratory conditions. The nitrogen fixed was quantified using the N isotopic dilution method. Variety BAS-370 had nearly 70% nitrogen derived from atmosphere (Ndfa) when inoculated with Azospirillum N-4. Similar studies with the mixed inoculum using N fertilizer in the micro plots indicated that nearly 29% of plant nitrogen was derived from the atmosphere.