Characterization of photorespiration in intact leaves using carbon dioxide labeling

The ¹³C nuclear magnetic resonance spectra of sucrose extracted from intact soybean and corn leaves labeled with ¹³CO₂ for 5 to 180 minutes have been obtained at 22.6 megahertz. The spectra provide a direct determination of the relative concentrations of ¹²C-¹³C and ¹³C-¹³C pairs of carbons in the labeled triose components of sucrose. This distribution of label is strongly dependent on the O₂ concentrations used during labeling for soybean but not for corn. Based on the time dependence of the experimental labeling patterns, photorespiration in air appears to be a sizable fraction of the apparent photosynthetic rate for soybean, but not for corn.     

Utilization of 13C-labeled amino acids to probe the α-helical local secondary structure of a membrane peptide using electron spin echo envelope modulation (ESEEM) spectroscopy

Electron spin echo envelope modulation (ESEEM) spectroscopy in combination with site-directed spin labeling (SDSL) has been established as a valuable biophysical technique to provide site-specific local secondary structure of membrane proteins. This pulsed electron paramagnetic resonance (EPR) method can successfully distinguish between α-helices, β-sheets, and 3₁₀-helices by strategically using ²H-labeled amino acids and SDSL. In this study, we have explored the use of ¹³C-labeled residues as the NMR active nuclei for this approach for the first time. ¹³C-labeled d₅-valine (Val) or ¹³C-labeled d₆-leucine (Leu) were substituted at a specific Val or Leu residue (i), and a nitroxide spin label was positioned 2 or 3 residues away (denoted i-2 and i-3) on the acetylcholine receptor M2δ (AChR M2δ) in a lipid bilayer. The ¹³C ESEEM peaks in the FT frequency domain data were observed for the i-3 samples, and no ¹³C peaks were observed in the i-2 samples. The resulting spectra were indicative of the α-helical local secondary structure of AChR M2δ in bicelles. This study provides more versatility and alternative options when using this ESEEM approach to study the more challenging recombinant membrane protein secondary structures.     

GmHKT1;4, a novel soybean gene regulating Na+/K+ ratio in roots enhances salt tolerance in transgenic plants

Salt is an important factor affecting the growth and development of soybean in saline or alkaline soil. The aims of the present study were to identify and functionally analyse the soybean GmHKTs gene family, and to explore their roles under NaHCO₃ and NaCl stresses. The GmHKTs gene family were isolated from soybean using genome sequence information. The GmHKTs gene family were further analysed for the structure and phylogenetic relationship. The expression patterns of soybean GmHKTs genes under NaHCO₃ and NaCl stresses were analysed via quantitative real-time PCR. As a result, the expression level of GmHKT1;4 was extremely up regulated in root under each treatment. Overexpression of GmHKT1;4 significantly enhanced the tolerance of transgenic tobacco plants to NaHCO₃ and NaCl stresses, compared with null plants. The overexpressed transgenic plants of this gene accomulated more K⁺ and less Na⁺ under salt stress, compaired with null plants. Our findings suggest that GmHKT1;4 plays an important role for regulation Na⁺/K⁺ ratio in roots under alkaline (NaHCO₃) and saline (NaCl) stresses.     

Multinuclear NMR of cis-dicarbonylbis(dithiocarbamato)iron(II) complexes in chloroform solution

The ¹³C and ¹⁵SN NMR spectra of eleven cis-Fe(S₂CNRR′)₂(CO)₂ complexes, where R and R′ are organic substituents, have been measured at ambient temperature in CDCl₃ (0.08–0.16 M). The ¹³C absorptions for the carbonyl ligands correlate well with the force constants for the CO stretching vibrations in CHCl₃ solution. Each of the parameters (¹³CO absorption and k_cis for CO) correlate well with the aqueous solution pK_a for⁺H₂NRR′, corrected for the phenyl-containing substituents, high pK_a values corresponding to high ¹³CO absorptions and low k_cis CO force constants. [p ]Evidence was found in the ¹³C NMR spectra for hindered rotation about the CN bond in S₂CNC₂ in complexes with higher pK_a(corr) values and in the ¹³C spectra of the corresponding thiuram disulfides. [p ]The ¹⁵N (natural abundance) NMR spectra for each of the complexes was determined. Each revealed a single sharp absorption in a region of the ¹⁵N NMR spectrum which indicates substantial CN double bond character, as one would expect for coordinated dithiocarbamate ligands.     

Kinetic complexities of triacylglycerol accumulation in developing embryos from Camelina sativa provide evidence for multiple biosynthetic systems

Quantitative flux maps describing glycerolipid synthesis can be important tools for rational engineering of lipid content and composition in oilseeds. Lipid accumulation in cultured embryos of Camelina sativa is known to mimic that of seeds in terms of rate of lipid synthesis and composition. To assess the kinetic complexity of the glycerolipid flux network, cultured embryos were incubated with [¹⁴C/¹³C]glycerol, and initial and steady state rates of [¹⁴C/¹³Cglyceryl] lipid accumulation were measured. At steady state, the linear accumulations of labeled lipid classes matched those expected from mass compositions. The system showed an apparently simple kinetic precursor–product relationship between the intermediate pool, dominated by diacylglycerol (DAG) and phosphatidylcholine (PC), and the triacylglycerol (TAG) product. We also conducted isotopomer analyses on hydrogenated lipid class species. [¹³C₃glyceryl] labeling of DAG and PC, together with estimates of endogenous [¹²C₃glyceryl] dilution, showed that each biosynthetically active lipid pool is ∼30% of the total by moles. This validates the concept that lipid sub-pools can describe lipid biosynthetic networks. By tracking the kinetics of [¹³C₃glyceryl] and [¹³C₂acyl] labeling, we observed two distinct TAG synthesis components. The major TAG synthesis flux (∼75%) was associated with >95% of the DAG/PC intermediate pool, with little glycerol being metabolized to fatty acids, and with little dilution from endogenous glycerol; a smaller flux exhibited converse characteristics. This kinetic heterogeneity was further explored using postlabeling embryo dissection and differential lipid extractions. The minor flux was tentatively localized to surface cells across the whole embryo. Such heterogeneity must be recognized in order to construct accurate gene expression patterns and metabolic networks describing lipid biosynthesis in developing embryos.     

Microsecond motions probed by near-rotary-resonance <Emphasis Type="Italic">R</Emphasis><Subscript>1ρ</Subscript><Superscript>15</Superscript>N MAS NMR experiments: the model case of protein overall-rocking in crystals

F_oF₁-ATP synthase catalyzes ATP hydrolysis/synthesis coupled with a transmembrane H⁺ translocation in membranes. The F_o c-subunit ring plays a major role in this reaction. We have developed an assignment strategy for solid-state ¹³C NMR (ssNMR) signals of the F_o c-subunit ring of thermophilic Bacillus PS3 (TF_o c-ring, 72 residues), carrying one of the basic folds of membrane proteins. In a ssNMR spectrum of uniformly ¹³C-labeled sample, the signal overlap has been a major bottleneck because most amino acid residues are hydrophobic. To overcome signal overlapping, we developed a method designated as COmplementary Sequential assignment with MInimum Labeling Ensemble (COSMILE). According to this method, we generated three kinds of reverse-labeled samples to suppress signal overlapping. To assign the carbon signals sequentially, two-dimensional C^α(i+1)–C′C^α(i) correlation and dipolar assisted rotational resonance (DARR) experiments were performed under magic-angle sample spinning. On the basis of inter- and intra-residue ¹³C–¹³C chemical shift correlations, 97% of C^α, 97% of C^β and 92% of C′ signals were assigned directly from the spectra. Secondary structure analysis predicted a hairpin fold of two helices with a central loop. The effects of saturated and unsaturated phosphatidylcholines on TF_o c-ring structure were examined. The DARR spectra at 15 ms mixing time are essentially similar to each other in saturated and unsaturated lipid membranes, suggesting that TF_o c-rings have similar structures under the different environments. The spectrum of the sample in saturated lipid membranes showed better resolution and structural stability in the gel state. The C-terminal helix was suggested to locate in the outer layer of the c-ring.     

3-Methoxyflavones and coumarins from Artemisia incanescens

Seven 3-methoxyflavones and three coumarins have been isolated from aerial parts of Artemisia incanescens. Their ¹H NMR spectra in DMSO-d₆ and CDCl₃ are compared and discussed. The hitherto unreported ¹³C NMR spectra of some of these compounds are also discussed.     

Structure,bonding and lattice dynamics of tri- and tetraphenyltin derivatives as studied by Mössbauer and multinuclear NMR spectroscopy

The structure and bonding properties of a number of closely related tetraphenyltin- and triphenyltin chloride compounds have been studied by the ¹¹⁹Sn Mössbauer effect and multinuclear NMR spectroscopy. The comparison of liquid and solid state ¹³C and ¹¹⁹Sn NMR spectra and of glassy solution matrix and neat solid state Mössbauer spectra provides information about the extent of intermolecular association effects in these compounds. The results indicate that all materials with the exception of (p-CF₃Ph)₃SnCl are adequately described as monomeric solids with tetrahedral geometry around the metal atom. For the latter compound spectroscopic evidence for the presence of a five-coordinated tin species is presented.     

The effect of the bilayer phase transition on the carbonyl carbon-13 chemical shift anisotropy

Proton enhanced (PE), natural abundance carbon-13 magnetic resonance spectra have been obtained of the carbonyl groups in hydrated dispersions of 1,2-dimyristoyl-sn-glycero-3-phosphocholine. A four-fold change in the overall linewidth results on passing from the fluid to crystalline phase. The carbonyl resonance provides a sensitive measure of the changes in mobility experienced by the lipid molecule above and below the phase transition temperature. The spectral shapes derived from both the fluid (T = 45°C) and crystalline (T = 15°C) phases indicate that even in the crystalline phase sufficient molecular motion is present to average the chemical shielding tensor. It is suggested that this motion in the L_β′ phase is a result of dislocations and packing faults diffusing in the plane of the bilayer. Because of the small size of the chemical shielding interaction (approx. 3 kHz for ω₀ = 22.63 MHz) lipid diffusion coefficients of order 10^?10 cm²/sec observed in the L_β′ phase [1] are effective in averaging the shielding tensor.A comparison is made with the perturbation suffered by the carbonyl groups in the L_α phase in the presence of substantial amounts of cholesterol or the polypeptide gramicidin A.     

Dynamics of Lipid Biosynthesis and Redistribution in the Marine Diatom Phaeodactylum tricornutum Under Nitrate Deprivation

One approach to achieve continuous overproduction of lipids in microalgal ??cell factories?? relies upon depletion or removal of nutrients that act as competing electron sinks (e.g., nitrate and sulfate). However, this strategy can only be effective for bioenergy applications if lipid is synthesized primarily de novo (from CO₂ fixation) rather than from the breakdown and interconversion of essential cellular components. In the marine diatom, Phaeodactylum tricornutum, it was determined, using ¹³C-bicarbonate, that cell growth in nitrate (NO ₃ ^? )-deprived cultures resulted predominantly in de novo lipid synthesis (60?% over 3?days), and this new lipid consisted primarily of triacylglycerides (TAGs). Nearly complete preservation of ¹²C occurred in all previously existing TAGs in NO ₃ ^? -deprived cultures and thus, further TAG accumulation would not be expected from inhibition of TAG lipolysis. In contrast, both high turnover and depletion of membrane lipids, phosphatidylcholines (PCs), were observed in NO ₃ ^? -deprived cultures (both the headgroups and fatty acid chains), while less turnover was observed in NO ₃ ^? replete cultures. Liquid chromatography-tandem mass spectrometry mass spectra and ¹³C labeling patterns of PC headgroups provided insight into lipid synthesis in marine diatoms, including suggestion of an internal pool of glycine betaine that feeds choline synthesis. It was also observed that 16C fatty acid chains incorporated into TAGs and PCs contained an average of 14 ¹³C carbons, indicating substantial incorporation of ¹³C-bicarbonate into fatty acid chains under both nutrient states.     

Dynamic structure of bombolitin II bound to lipid bilayers as revealed by solid-state NMR and molecular-dynamics simulation

Bombolitin II (BLT2) is one of the hemolytic heptadecapeptides originally isolated from the venom of a bumblebee. Structure and orientation of BLT2 bound to 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) membranes were determined by solid-state ³¹P and ¹³C NMR spectroscopy. ³¹P NMR spectra showed that BLT2-DPPC membranes were disrupted into small particles below the gel-to-liquid crystalline phase transition temperature (T_c) and fused to form a magnetically oriented vesicle system where the membrane surface is parallel to the magnetic fields above the T_c. ¹³C NMR spectra of site-specifically ¹³C-labeled BLT2 at the carbonyl carbons were observed and the chemical shift anisotropies were analyzed to determine the dynamic structure of BLT2 bound to the magnetically oriented vesicle system. It was revealed that the membrane-bound BLT2 adopted an α-helical structure, rotating around the membrane normal with the tilt angle of the helical axis at 33°. Interatomic distances obtained from rotational-echo double-resonance experiments further showed that BLT2 adopted a straight α-helical structure. Molecular dynamics simulation performed in the BLT2-DPPC membrane system showed that the BLT2 formed a straight α-helix and that the C-terminus was inserted into the membrane. The α-helical axis is tilted 30° to the membrane normal, which is almost the same as the value obtained from solid-state NMR. These results suggest that the membrane disruption induced by BLT2 is attributed to insertion of BLT2 into the lipid bilayers.     

The effect of acidification and the combined effects of acidification/lipid extraction on carbon stable isotope ratios for sub-arctic and arctic marine zooplankton species

Stable isotope ratios of carbon (δ ¹³C) in zooplankton are widely applied in ecosystem-level studies of energy flux and trophic interactions. Carbonate (CaCO₃) and lipid content are highly variable both among and within zooplankton species. Such variability can arise from the δ ¹³C-depleted nature of lipids as well as differences in carbon incorporation among tissues (e.g., relative amount of carbonate). Critically, the impact of the common lipid- and carbonate-normalization steps of extraction and acidification is poorly understood and applied in an inconsistent manner. Here, we investigated the effect of lipid extraction and sample acidification (CaCO₃ removal) on δ ¹³C in sub-arctic and arctic marine zooplankton species. Our results indicate that, with the exception of the shelled mollusc Limacina helicina, acidification of samples can be omitted for all other marine zooplankton considered in this study. In the case of L. helicina, δ ¹³C can be corrected for carbonate content using the linear equation developed in this study. In contrast, the δ ¹³C for all species was significantly enriched by a combination of lipid extraction and acidification (up to +4.9 ‰) prior to stable isotope analysis. Our data were used to develop simple, predictive species-specific correction models for lipid-acid-normalized δ ¹³C using C:N and/or untreated δ ¹³C values. Our results indicate that the δ ¹³C value for all species, including those with lower C:N ratios (~3–4), should be corrected for lipid content. We recommend lipid extraction whenever possible, or else the use of species/taxon-specific δ ¹³C lipid-normalization models for accurate determination of carbon sources and dynamics for arctic and sub-arctic marine zooplankton.     

Étude par r.m.n. de quatre disaccharides peracétylés sélectivement enrichis en 13C

Four peracetylated disaccharides ¹³C-labelled at the C-1′ position and having α-d-(1′→3), β-d-(1′→3), α-d-(1′→4), and β-d-(1′→4) linkages were prepared starting from the commercially available d-[1-¹³C]glucose. They were studied on the basis of their ³J_¹³CH coupling constants in relation with the conformation in solution of oligosaccharides as models for the corresponding polymer. A method of analysis of the n.m.r. spectra is described and the coupling constants J_{¹³C-1′H} given, particularly the ²J coupling (in the same cycle and with sign determination) and the ³J coupling (through the glycosidic bond). In that case, the values obtained give experimental information on the ψ angle values. They are compared with the known X-ray data for similar compounds.     

Utilization of 13C-13C Coupling in Structural and Biosynthetic Studies Double Labeling Method

A new method which utilizes ¹³C-¹³C coupling for structural and biosynthetic studies on acetate-derived metabolites is described. The ¹³C-NMR spectra of dihydrolatumcidins separately labeled with ¹³CH₃¹³C0₂Na and with a 1: 1 mixture of ¹³CH₃CO₂-Na and CH313C02-Na gave enough information to establish its structure.     

The membrane-induced structure of melittin is correlated with the fluidity of the lipids

The effect of the bee toxin melittin on DMPC dynamics in fast-tumbling bicelles has been investigated. The ¹³C R₁ and ¹³C-¹H NOE relaxation parameters for DMPC were used to monitor the effect of melittin and cholesterol on lipid dynamics. It was found that melittin has the largest effect on the DMPC mobility in DMPC/DHPC bicelles, while less effect was observed in cholesterol-doped bicelles, or in bicelles made with CHAPS, indicating that the rigidity of the membrane affects the melittin-membrane interaction. CD spectra were analysed in terms of cooperativity of the α-helix to random coil transition in melittin, and these results also indicated similar differences between the bicelles. The study shows that bicelles can be used to investigate lipid dynamics by spin relaxation, and in particular of peptide-induced changes in membrane fluidity.     

Evidence of pores and thinned lipid bilayers induced in oriented lipid membranes interacting with the antimicrobial peptides, magainin-2 and aurein-3.3

Dynamic structures of supramolecular lipid assemblies, such as toroidal pores and thinned bilayers induced in oriented lipid membranes, which are interacting with membrane-acting antimicrobial peptides (AMPs), magainin-2 and aurein-3.3, were explored by ³¹P and ²H solid-state NMR (ssNMR) spectroscopy. Various types of phospholipid systems, such as POPC-d₃₁, POPC-d₃₁/POPG, and POPC-d₃₁/cholesterol, were investigated to understand the membrane disruption mechanisms of magainin-2 and aurein-3.3 peptides at various peptide-to-lipid (P:L) ratios. The experimental lineshapes of anisotropic ³¹P and ²H ssNMR spectra measured on these peptide-lipid systems were simulated reasonably well by assuming the presence of supramolecular lipid assemblies, such as toroidal pores and thinned bilayers, in membranes. Furthermore, the observed decrease in the anisotropic frequency span of either ³¹P or ²H ssNMR spectra of oriented lipid bilayers, particularly when anionic POPG lipids are interacting with AMPs at high P:L ratios, can directly be explained by a thinned membrane surface model with fast lateral diffusive motions of lipids. The spectral analysis protocol we developed enables extraction of the lateral diffusion coefficients of lipids distributed on the curved surfaces of pores and thinned bilayers on a few nanometers scale.     

Synthesis of oligomethylene-strapped chlorophyll derivatives and optical properties of their stereoisomers in a solution

Methyl pheophorbide-a/a′ derivatives covalently linked with oligomethylene chains at the 3-CH₂OCO– and 13²-COO– moieties in a molecule were prepared by modifying chlorophyll-a through intramolecular ring-closing metathesis of vinyl groups. At least, a C10-length between the 3³- and 13⁴-positions was necessary for the cyclization and connection of a C12-strap was the most suitable to achieve the highest closure yield. The oligomethylene chain in 13² R-epimers derived from methyl pheophorbide-a covered the α-face of the chlorin π-plane and the strap in the corresponding 13² S-epimers protected the β-face. Synthetic 13² R-epimer with a dodecamethylene chain gave a flat chlorin π-plane, while the decamethylene chain in the 13² R-epimer distorted the π-system due to its shorter linkage. The distortion by strapping in the 13² R-epimer induced a slight blue-shift of Qy peak in dichloromethane. CD spectra of the 13² R-epimers were similarly dependent on the chain length, i.e., the distortion of π-plane. Visible absorption and CD spectra of all the strapped 13² S-epimers were almost identical and only slightly different from those of the unstrapped. The strapping in the 13² S-epimers shifted the Qy peak bathochromically.     

Initial Organic Products of Fixation of [N]Dinitrogen by Root Nodules of Soybean (Glycine max)

When detached soybean Glycine max (L.) Merr. cv. Hark, nodules assimilate [¹³N]N₂, the initial organic product of fixation is glutamine; glutamate becomes more highly radioactive than glutamine within 1 minute; ¹³N in alanine becoms detectable at 1 minute of fixation and increases rapidly between 1 and 2 minutes. After 15 minutes of fixation, the major ¹³N-labeled organic products in both detached and attached nodules are glutamate and alanine, plus, in the case of attached nodules, an unidentified substance, whereas [¹³N]glutamine comprises only a small fraction of organic ¹³N, and very little ¹³N is detected in asparagine. The fixation of [¹³N]N₂ into organic products was inhibited more than 99% by C₂H₂ (10%, v/v). The results support the idea that the glutamine synthetase-glutamate synthase pathway is the primary route for assimilation of fixed nitrogen in soybean nodules.     

A high-resolution NMR study (1H, 13C, 31P) of the interaction of paramagnetic ions with phospholipids in aqueous dispersions

¹H-, ¹³C-and ³¹P-NMR spectra of egg-yolk phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidic acid (PA) and cosonicated mixtures of these phospholipids were obtained from ultrasonicatcd dispersions containing Pr³⁺, Eu³⁺, Gd³⁺ and Mn²⁺ ions.The differences in chemical shift values. °_n, between the “inner” and “outer” resonance signals for the different nuclei of the polar head group of egg-yolk phosphatidyl choline provide information about the average distances of the paramagnetic ion within the polar groups of the phospholipid molecules. In the Pr(²H₂O)³⁺_n/egg-yolk phosphatidylcholine system the ions are nearest to the phosphate and -CH₂CH₂ group, respectively but relatively far from the N(CH₃)₃ group of the polar head group of the lipid.The integral analysis of the¹ H-NMR spectra obtained from dispersions containing Pr³⁺ and Mn²⁺ ions enables us to calculate the number of the polar groups in both sides of the egg-yolk phosphatidylcholine bilayer, the size of the lipid vesicle and to give some features of the arrangement of the phospholipid molecules in cosonicated egg-yolk phosphatidylcliotine/ phosphatidytserine vesicles. At p²H 8.3 in PC/PS mixtures an extreme asymmetry is observed with PS preferentially in the outer side of the membrane. This side contains approximately three times more PS than PC molecules.Some comments are made concerning the quantitative integral analysis of proton-noise decoupled ³¹ P-NMR spectra as obtained from similar phospholipid mixtures by Michaelson et al. and Berden et at.     

Lipid Polymorphism Induced by Surfactant Peptide SP-B1-25

Pulmonary surfactant protein B (SP-B) is an essential protein for lowering surface tension in the alveoli. SP-B_1-25, a peptide comprised of the N-terminal 25 amino-acid residues of SP-B, is known to retain much of the biological activity of SP-B. Circular dichroism has shown that when SP-B_1-25 interacts with negatively charged lipid vesicles, it contains significant helical structure for the lipid compositions and peptide/lipid ratios studied here. The effect of SP-B_1-25 on lipid organization and polymorphisms was investigated via DSC, dynamic light scattering, transmission electron microscopy, and solid-state NMR spectroscopy. At 1-3 mol% peptide and physiologic temperature, SP-B_1-25 partitions at the interface of negatively charged PC/PG lipid bilayers. In lipid mixtures containing 1-5 mol% peptide, the structure of SP-B_1-25 remains constant, but ²H and ³¹P NMR spectra show the presence of an isotropic lipid phase in exchange with the lamellar phase below the T_m of the lipids. This behavior is observed for both DPPC/POPG and POPC/POPG lipid mixtures as well as for both the PC and PG components of the mixtures. For 1-3 mol% SP-B_1-25, a return to a single lamellar phase above the lipid mixture T_m is observed, but for 5 mol% SP-B_1-25 a significant isotropic component is observed at physiologic temperatures for DPPC and exchange broadening is observed in ²H and ³¹P NMR spectra of the other lipid components in the two mixtures. DLS and TEM rule out the formation of micellar structures and suggest that SP-B_1-25 promotes the formation of a fluid isotropic phase. The ability of SP-B_1-25 to fuse lipid lamellae via this mechanism, particularly those enriched in DPPC, suggests a specific role for the highly conserved N-terminus of SP-B in the packing of lipid lamellae into surfactant lamellar bodies or in stabilizing multilayer structures at the air-liquid interface. Importantly, this behavior has not been seen for the other SP-B fragments of SP-B_8-25 and SP-B_59-80, indicating a critical role for the proline rich first seven amino acids in this protein.