Stable Water Use Efficiency of Tibetan Alpine Meadows in Past Half Century: Evidence from Wool δ13C Values

Understanding the influences of climatic changes on water use efficiency (WUE) of Tibetan alpine meadows is important for predicting their long-term net primary productivity (NPP) because they are considered very sensitive to climate change. Here, we collected wool materials produced from 1962 to 2010 and investigated the long-term WUE of an alpine meadow in Tibet on basis of the carbon isotope values of vegetation (δ ¹³C_veg). The values of δ ¹³C_veg decreased by 1.34‰ during 1962–2010, similar to changes in δ ¹³C values of atmospheric CO₂. Carbon isotope discrimination was highly variable and no trend was apparent in the past half century. Intrinsic water use efficiency (W _i) increased by 18 μmol·mol^–1 (approximately 23.5%) during 1962–2010 because the increase in the intercellular CO₂ concentration (46 μmol·mol^–1) was less than that in the atmospheric CO₂ concentration (C _a, 73 μmol·mol^–1). In addition, W _i increased significantly with increasing growing season temperature and C _a. However, effective water use efficiency (W _e) remained relatively stable, because of increasing vapor pressure deficit. C _a, precipitation, and growing season temperature collectively explained 45% of the variation of W _e. Our findings indicate that the W _e of alpine meadows in the Tibetan Plateau remained relatively stable by physiological adjustment to elevated C _a and growing season temperature. These findings improve our understanding and the capacity to predict NPP of these ecosystems under global change scenarios.     

Determination of 13Cα relaxation times in uniformly 13C/15N-enriched proteins

Summary Relaxation times of ¹³C carbons of uniformly ¹³C/¹⁵N-enriched probes have been investigated. The relaxation behaviour was analyzed in terms of a multispin system. Pulse sequences for the determination of T₁, T₂ and the heteronuclear NOE of ¹³C in uniformly ¹³C/¹⁵N-enriched ribonuclease T1 are presented. The experiments performed in order to obtain T₁ and the heteronuclear NOE were similar to those of the corresponding ¹⁵N experiments published previously. The determination of T₂ for the C-carbon in a completely labeled protein is more complicated, since the magnetization transfer during the T₂ evolution period owing to the scalar coupling of C–C must be suppressed. Various different pulse sequences for the T₂ evolution period were simulated in order to optimize the bandwidth for which reliable T₂ relaxation times can be obtained. A proof for the quality of these pulse sequences is given by fitting the intensity decay of individual ¹H–¹³C cross peaks, in a series of (¹H, ¹³C)-ct-HSQC spectra with a modified CPMG sequence as well as a T_1p sequence for the transverse relaxation time, to a single exponential using a simplex algorithm.     

Stratification of δ13C values of leaves in Amazonian rain forests

Summary The contribution of soil respiration to the photosynthesis of the shade flora in the Amazon forest was evaluated by measuring the ¹³C values of leaves collected at different levels in two forest communities. Canopy leaves have an average ¹³C of-30.5 in the podsol forest and-28.7 in the laterite forest. Leaves from plants in the lower forest strata have a significantly lower value of-35.2 in the podsol forest and-34.3 in the laterite forest.Mailing address of the first author: Before May 31, 1980: Department of Biological Sciences, Stanford University, Stanford, California 94305 USA. After May 31: Centro de Ecologia, IVIC Aptdo. 1827. Caracas, Venezuela     

Trimethylamine and Organic Matter Additions Reverse Substrate Limitation Effects on the δ13C Values of Methane Produced in Hypersaline Microbial Mats

Methane production has been observed in a number of hypersaline environments, and it is generally thought that this methane is produced through the use of noncompetitive substrates, such as the methylamines, dimethylsulfide and methanol. Stable isotope measurements of the produced methane have also suggested that the methanogens are operating under conditions of substrate limitation. Here, substrate limitation in gypsum-hosted endoevaporite and soft-mat hypersaline environments was investigated by the addition of trimethylamine, a noncompetitive substrate for methanogenesis, and dried microbial mat, a source of natural organic matter. The δ¹³C values of the methane produced after amendments were compared to those in unamended control vials. At all hypersaline sites investigated, the δ¹³C values of the methane produced in the amended vials were statistically lower (by 10 to 71‰) than the unamended controls, supporting the hypothesis of substrate limitation at these sites. When substrates were added to the incubation vials, the methanogens within the vials fractionated carbon isotopes to a greater degree, resulting in the production of more ¹³C-depleted methane. Trimethylamine-amended samples produced lower methane δ¹³C values than the mat-amended samples. This difference in the δ¹³C values between the two types of amendments could be due to differences in isotope fractionation associated with the dominant methane production pathway (or substrate used) within the vials, with trimethylamine being the main substrate used in the trimethylamine-amended vials. It is hypothesized that increased natural organic matter in the mat-amended vials would increase fermentation rates, leading to higher H₂ concentrations and increased CO₂/H₂ methanogenesis.     

Effect of C3–C4 Vegetation Change on δ13C and δ15N Values of Soil Organic Matter Fractions Separated by Thermal Stability

Carbon isotopic composition of soils subjected to C₃–C₄ vegetation change can be used to estimate C turnover in bulk soil and in soil organic matter (SOM) pools with fast and intermediate turnover rates. We hypothesized that the biological availability of SOM pools is inversely proportional to their thermal stability, so that thermogravimetry can be used to separate SOM pools with contrasting turnover rates. Soil samples from a field plot cultivated for 10.5 years with the perennial C₄ plant Miscanthus×gigantheus were analyzed by thermogravimetry coupled with differential scanning calorimetry (DSC). Three SOM fractions were distinguished according to the differential weight losses and exothermic or endothermic reactions measured by DSC. The δ¹³C and δ¹⁵N values of these three fractions obtained by gradual soil heating were measured by IRMS. The weight losses up to 190 °C mainly reflected water evaporation because no significant C and N losses were detected and δ¹³C and δ¹⁵N values of the residual SOM remained unchanged. The δ¹³C values (−16.4‰) of SOM fraction decomposed between 190 and 390 °C (containing 79% of total soil C) were slightly closer to that of the Miscanthus plant tissues (δ¹³C = −11.8‰) compared to the δ¹³C values (−16.8‰) of SOM fraction decomposed above 390 °C containing the residual 21% of SOM. Thus, the C turnover in the thermally labile fraction was faster than that in thermally stable fractions, but the differences were not very strong. Therefore, in this first study combining TG-DSC with isotopic analysis, we conclude that the thermal stability of SOM was not very strongly related to biological availability of SOM fractions. In contrast to δ¹³C, the δ¹⁵N values strongly differed between SOM fractions, suggesting that N turnover in the soil was different from C turnover. More detailed fractionation of SOM by thermal analysis with subsequent isotopic analysis may improve the resolution for δ¹³C.     

Measurement of 13Cα-13CO cross-relaxation rates in 15N-/13C-labelled proteins

Summary Internal motions play an important role in the biological function of proteins and NMR relaxation studies may characterize them over a wide range of frequencies. An experimental pulse scheme is proposed for the measurement of the ¹³CO-¹³C cross-relaxation rate. For sensitivity reasons, this measurement is performed in an indirect manner through several coherence transfer steps, which should thus be calibrated independently. Contributions of other relaxation pathways can be eliminated by the determination of the initial slope of the buildup curve. The cross-relaxation rates have been determined on a ¹⁵N-/¹³C-labelled 116-residue protein and the significant variations along the sequence have been interpreted as evidence of an increased amount of fast local motion.     

Study of protein dynamics in solution by measurement of 13Cα-13CO NOE and 13CO longitudinal relaxation

Summary ¹³C-¹³CO homonuclear NOE and ¹³CO T₁ relaxation were measured for a 20 kDa protein using tripleresonance pulse sequences. The experiments were sufficiently sensitive to obtain statistically significant differences in relaxation parameters over the molecule. The ¹³C-¹³CO cross-relaxation rate, obtained from these data, is directly proportional to an order parameter describing local motion and it is largely independent of the local correlation time. It is therefore a relatively straightforward observable for the identification of local dynamics.     

Automated prediction of 15N, 13Cα, 13Cβ and 13C′ chemical shifts in proteins using a density functional database

A database of peptide chemical shifts, computed at the density functional level, has been used to develop an algorithm for prediction of ¹⁵N and ¹³C shifts in proteins from their structure; the method is incorporated into a program called SHIFTS (version 4.0). The database was built from the calculated chemical shift patterns of 1335 peptides whose backbone torsion angles are limited to areas of the Ramachandran map around helical and sheet configurations. For each tripeptide in these regions of regular secondary structure (which constitute about 40% of residues in globular proteins) SHIFTS also consults the database for information about sidechain torsion angle effects for the residue of interest and for the preceding residue, and estimates hydrogen bonding effects through an empirical formula that is also based on density functional calculations on peptides. The program optionally searches for alternate side-chain torsion angles that could significantly improve agreement between calculated and observed shifts. The application of the program on 20 proteins shows good consistency with experimental data, with correlation coefficients of 0.92, 0.98, 0.99 and 0.90 and r.m.s. deviations of 1.94, 0.97, 1.05, and 1.08 ppm for ¹⁵N, ¹³C, ¹³C and ¹³C, respectively. Reference shifts fit to protein data are in good agreement with `random-coil' values derived from experimental measurements on peptides. This prediction algorithm should be helpful in NMR assignment, crystal and solution structure comparison, and structure refinement.     

Quantitative J correlation methods for the accurate measurement of 13C′-13Cαdipolar couplings in proteins

Methods are described for the precise and accurate measurement of one-bond dipolar (13)C'-(13)C(alpha) couplings in weakly aligned proteins. The experiments are based on the principle of quantitative J correlation, where (1)J(C'C(alpha)) (or (1)J(C'C(alpha)) + 1D(C'C(alpha)) is measured from the relative intensity of two interleaved 3D TROSY-HN(CO)CA or 3DTROSY-HNCO spectra recorded with dephasing intervals of zero (reference spectrum) and approximately 3/(2(1)J(C'C(alpha)) (attenuated spectrum). In analogy to other quantitative J correlation techniques, the random error in the measured (1)J(C'C(alpha)) value is inversely proportional to the signal-to-noise ratio in the reference spectrum. It is shown that for weakly aligned proteins, with the magnitude of the alignment tensor of D(a)(NH) < or = 10-15 Hz, the systematic errors are typically negligible. The methods are demonstrated for the third IgG-binding domain of protein G (GB3) and a-synuclein in complex with a detergent micelle, where errors in (1)D(C'C(alpha)) of less than 0.1 Hz and ca. 0.2 Hz,respectively, are estimated. Remarkably, the dipolar couplings determined for GB3 are in even better agreement with the recently refined 1.1-angstroms X-ray structure than the input (13)C'-(13)C(alpha) couplings used for the refinement.     

δ13C values of an herbivore and the ratio of C3 to C4 plant carbon in its diet

Summary The ¹³C values of whole body samples of the beetle Tribolium castaneum are closely correlated with the ¹³C values of the plant carbon in its diet. The correlation is always high for diets ranging from 100% C₄ to 100% C₃ plant material. The degree of correlation is independent of the growth rate of the animals.     

Interpretations of gradients in δ13C value in thick photosynthetic tissues of plants with Crassulacean acid metabolism

In Ceropegia dichotoma, Crassula argentea, Esheveria colorata, Kalanchoë beharensis, Opuntia ficus-indica, Sansveria stuckyi and Opuntia inermis the carbon-isotope ratio ( ¹³C) of tissues close to the epidermis is 2–4.3 more negative than those in the centre of the leaf or cladode. The greatest change in ¹³C value occurs between the epidermal layer and the layer of mesophyll tissue immediately underneath. Analysis of major metabolic and structural components in successive layers of Crassula argentea grown under controlled environmental conditions conducive to Crassulacean acid metabolism confirmed that much of the variation in ¹³C values of bulk carbon is caused by differences in chemical composition. Thus the steep gradient in ¹³C value at the epidermis reflects, in part, the contribution of more-negative ¹³C values of lipids in these tissues. Moreover, during nocturnal CO₂ fixation the amount of malic acid synthesised decreases with depth and the ¹³C value of the methanol-soluble fraction is less negative with distance away from the upper epidermis. These results are consistent with diffusion limitation to CO₂ uptake in these thick leaf tissues, which also contributes to the observed gradients in ¹³C value.Abbreviation CAM Crassulacean acid metabolism - FW fresh weight - PEPCase phospoenolpyruvate carboxylase - Rubisco ribulose-1,5-bisphosphate carboxylase-oxygenase - ¹³C carbon-isotope ratio This work was supported by grants from the North Carolina Biotechnology Centre to Duke University, National Science Foundation (NSF) grant DCB90-06830, Department of Energy, Division of Energy Biosciences grant DE-FG05-89 ER 14005, and NSF grant BSR 87-06429 to Duke University Phytotron.     

The 13C/12C isotopic signal of day-respired CO2 in variegated leaves of Pelargonium × hortorum

In leaves, although it is accepted that CO(2) evolved by dark respiration after illumination is naturally (13) C-enriched compared to organic matter or substrate sucrose, much uncertainty remains on whether day respiration produces (13) C-depleted or (13) C-enriched CO(2). Here, we applied equations described previously for mesocosm CO(2) exchange to investigate the carbon isotope composition of CO(2) respired by autotrophic and heterotrophic tissues of Pelargonium × hortorum leaves, taking advantage of leaf variegation. Day-respired CO(2) was slightly (13) C-depleted compared to organic matter both under 21% O(2) and 2% O(2). Furthermore, most, if not all CO(2) molecules evolved in the light came from carbon atoms that had been fixed previously before the experiments, in both variegated and green leaves. We conclude that the usual definition of day respiratory fractionation, that assumes carbon fixed by current net photosynthesis is the respiratory substrate, is not valid in Pelargonium leaves under our conditions. In variegated leaves, total organic matter was slightly (13) C-depleted in white areas and so were most primary metabolites. This small isotopic difference between white and green areas probably came from the small contribution of photosynthetic CO(2) refixation and the specific nitrogen metabolism in white leaf areas.     

δ13C values of grass species collected in the northern Sahara desert

Summary ¹³C values were measured for 45 Poaceae species collected in the northern Sahara desert, at the foot of the Saharan Atlas. The results indicate a clear relationship between carbon isotope discrimination and phytogeographical distribution of the grasses. Mediterranean species predominantly had ¹³C values indicating the C₃ pathway of photosynthesis. By contrast, nearly all species belonging to the Saharo-Arabian and /or Sudanian group showed a C₄ like carbon isotope composition. Leaf material of two species, Lygeum spartum and Stipa tenacissima, had ¹³C values in the region of-20, i.e. intermediate between the mean ¹³C values of C₃ and C₄ plants. However, additional speciments of both these grasses obtained from a different source (herbarium of the Hebrew University, Jerusalem) yielded a C₃ like carbon isotope composition.     

A 13C NMR spectrometric method for the determination of intramolecular δ13C values in fructose from plant sucrose samples

Recent developments in (13) C NMR spectrometry have allowed the determination of intramolecular (13) C/(12) C ratios with high precision. However, the analysis of carbohydrates requires their derivatization to constrain the anomeric carbon. Fructose has proved to be particularly problematic because of a byproduct occurring during derivatization and the complexity of the NMR spectrum of the derivative. Here, we describe a method to determine the intramolecular (13) C/(12) C ratios in fructose by (13) C NMR analysis of the acetyl-isopropylidene derivative. We have applied this method to measure the intramolecular (13) C/(12) C distribution in the fructosyl moiety of sucrose and have compared this with that in the glucosyl moiety. Three prominent features stand out. First, in sucrose from both C(3) and C(4) plants, the C-1 and C-2 positions of the glucosyl and fructosyl moieties are markedly different. Second, these positions in C(3) and C(4) plants show a similar profile. Third, the glucosyl and fructosyl moieties of sucrose from Crassulacean acid metabolism (CAM) metabolism have a different profile. These contrasting values can be interpreted as a result of the isotopic selectivity of enzymes that break or make covalent bonds in glucose metabolism, whereas the distinctive (13) C pattern in CAM sucrose probably indicates a substantial contribution of gluconeogenesis to glucose synthesis.     

Determination of the 13C chemical-shift tensors in a single crystal of methyl α-d-glucopyranoside.

The chemical shielding tensors and their direction cosines of the ¹³C nuclei in a single crystal of methyl α-d-glucopyranoside were determined by the high-resolution solid state n.m.r. technique. The results were used to assign the ¹³C cross-polarization, magic angle spinning (c.p.-m.a.s.) spectrum of a polycrystalline sample of that compound. The differences between the ¹³C chemical shifts observed in the c.p.-m.a.s. spectrum of the solid and of solutions of methyl α-d-glucopyranoside are discussed in terms of the different types of hydrogen bonds formed in the crystalline state and in solution.     

13C–13C NOESY spectra of a 480 kDa protein: solution NMR of ferritin

Molecular size has limited solution NMR analyses of proteins. We report ¹³C–¹³C NOESY experiments on a 480 kDa protein, the multi-subunit ferritin nanocage with gated pores. By exploiting ¹³C-resonance-specific chemical shifts and spin diffusion effects, we identified 75% of the amino acids, with intraresidue C–C connectivities between nuclei separated by 1–4 bonds. These results show the potential of ¹³C–¹³C NOESY for solution studies of molecular assemblies >100 kDa.     

Analysis of δ13C of CO2 distinguishes between microbial respiration of added C4-sucrose and other soil respiration in a C3-ecosystem

The main aim of this study was to test various hypotheses regarding the changes in ¹³C of emitted CO₂ that follow the addition of C₄-sucrose to the soil of a C₃-ecosystem. It forms part of an experimental series designed to assess whether or not the contributions from C₃-respiration (root and microbial) and C₄-respiration (microbial) to total soil respiration can be calculated from such changes. A series of five experiments, three on sieved (root-free) mor-layer material, and two in the field with intact mor-layer (and consequently with active roots), were performed. Both in the experiments on sieved mor-layer and the field experiments, we found a C₄-sucrose-induced increase in C₃-respiration that accounted for between 30% and 40% of the respiration increase 1 h after sucrose addition. When the course of C₃-, C₄- and total respiration was followed in sieved material over four days following addition of C₄-sucrose, the initially increased respiration of C₃-C was transient, passing within less than 24 h. In a separate pot experiment, neither ectomycorrhizal Pinus sylvestrisL. roots nor non-mycorrhizal roots of this species showed respiratory changes in response to exogenous sucrose. No shift in the ¹³C of the evolved CO₂ after adding C₃-sucrose to sieved mor-layer material was found, confirming that the sucrose-induced increase in respiration of endogenous C was not an artefact of discrimination against ¹³C during respiration. Furthermore, we conclude that the C₄-sucrose induced transient increase in C₃-respiration is most likely the result of accelerated turnover of C in the microbial biomass. Thus, neither respiration of mycorrhizal roots, nor processes discriminating against ¹³C were likely sources of error in the field. The estimated ¹³C of evolved soil CO₂ in three field experiments lay between –25.2 and –23.6. The study shows that we can distinguish between CO₂ evolved from microbial mineralisation of added C₄-sucrose, and CO₂ evolved from endogenous carbon sources (roots and microbial respiration).     

Variation in foliar δ13C in Hawaiian Metrosideros polymorpha: a case of internal resistance?

Summary Sun leaves of Metrosideros polymorpha were collected in 51 sites on 9 lava flows that represented gradients of elevation, precipitation, substrate age, and substrate texture on Mauna Loa volcano, Hawai'i. Leaf mass per unit leaf area increased with increasing elevation on all flows, while foliar nitrogen concentration decreased with increasing elevation and increased with increasing substrate age. Foliar ¹³C became less negative with increasing elevation on the wet east-side lava flows, but not the dry northwest-side flows; it did not reflect patterns of precipitation or presumed water availability. ¹³C was very strongly correlated with leaf mass per area across all of the sites. Limited gas-exchange information suggested that calculated c_i/c_a did not decrease with elevation in association with less-negative ¹³C, and photosynthesis per unit of nitrogen was significantly reduced in high-elevation plants. These results are consistent with a substantial internal resistance to CO₂ diffusion in the thick Metrosideros polymorpha leaves in high elevation sites.Published as a contribution to the International Union for Biological Sciences Tropical Mountain Ecosystems Program, and as Carnegie Institution of Washington Department of Plant Biology Publication Number 1065     

Changes in δ 13C stable isotopes in multiple tissues of insect predators fed isotopically distinct prey

Traditionally, researchers have used measurements of carbon stable isotopes to infer the composition of consumers’ diets. However, since consumer’s tissues may process carbon isotopes differently, particularly following a diet shift, it is possible to use measurements of carbon isotopes in multiple tissues to determine not only the composition of an individual’s diet, but also the temporal dynamics thereof. This study examined how stable isotopes of carbon (¹³C/¹²C, expressed as δ ¹³C) changed in different adult tissues of two predacious beetles, Harmonia axyridis and Coccinella septempunctata (Coleoptera: Coccinellidae). In the laboratory, we switched ladybeetles from a C3-based diet (soybean aphids, Aphis glycines) to a C4-based one (corn leaf aphids, Rhopalosiphum maidis). The δ ¹³C of metabolically active tissues such as the body fat and reproductive organs changed rapidly (≤5 days) following the diet shift. Tissues expected to be more metabolically inert, such as wings, changed more slowly over the same period. Although these general patterns were largely similar between males and females, females had more rapid changes in δ ¹³C in fat and reproductive tissues. However, females showed a significant depletion in δ ¹³C after 10 days, while males’ δ ¹³C continued to increase. Given the results of this experiment, it is now possible to distinguish between ladybeetles eating a mixed diet (beetles with multiple tissues at similar, intermediate, equilibrial δ ¹³C signatures) from those that have shifted diets (beetles with different tissues at distinctly different δ ¹³C values). Thus, this approach can be used broadly to infer not only what constitutes the diet of a consumer, but also the temporal history of dietary intake.