Variation in bulk tissue, fatty acid and monosaccharide δ13C values between autotrophic and heterotrophic plant organs

The flowers of 23 species of grass and herb plants were collected from a mesotrophic grassland to assess natural variability in bulk, monosaccharide and fatty acid δ¹³C values from one plant community and were compared with previous analyses of leaves from the same species. The total mean bulk δ¹³C value of flower tissues was −28.1‰, and there was no significant difference between the mean δ¹³C_flower values for grass (−27.8‰) and herb (−28.2‰) species. On average bulk δ¹³C_flower values were 1.1‰ higher than bulk δ¹³C_leaf values, however, the δ¹³C_flower and δ¹³C_leaf values of grasses did not differ between organs suggesting that carbon isotope discrimination is different in grass and herb species. The abundance of different monosaccharides abundance varied between plant types, i.e. xylose concentrations in the grass flowers were as high as 40%, compared with up to 15% in the herb species, but the general relationship δ¹³C_arabinose > δ¹³C_xylose > δ¹³C_glucose > δ¹³C_galactose which had been observed in leaves was similar in flowers (total mean δ¹³C values = −25.9‰, −27.2‰, −28.8‰ and −28.1‰, respectively). However, the average 5.4‰ depletion in the δ¹³C values of the C_16:0, C_18:2 and C_18:3 fatty acids in flowers compared to bulk tissue was significantly greater than observed for leaves. The trend C_16:0 < C_18:2 < C_18:3 previously observed in leaves was also observed in grass flowers (δ¹³C_C16:0 = −33.8‰; δ¹³C_C18:2 = −33.1‰; δ¹³C_C18:3 = −34.2‰) but not herb flowers (δ¹³C_C16:0 = −34.1‰; δ¹³C_C18:2 = −32.4‰; δ¹³C_C18:3 = −34.5‰). We conclude: (i) that the biological processes influencing carbon isotope discrimination in grass flowers are different from herbs flowers; and, (ii) that a range of post-photosynthetic fractionation effects caused the observed differences between flower and leaf δ¹³C values, especially the significant ¹³C-depletion in flower fatty acid δ¹³C values.     

An NMR study of the loss of carbon-20 in the biosynthesis of gibberellin A3 by Gibberella fujikuroi

Resuspension cultures of Gibberella fujikuroi, strain GF-1a, were shown to metabolise potassium [3′-¹³C] mevalonate to ¹³C-enriched C₁₉-gibberellins, plus ¹³CO₂ (derived from the loss of carbon-20). The formation of [¹³C]-gibberellins could be observed in vivo using ¹³C NMR; however that of ¹³CO₂ could not. In contrast, removal of the mycelium and concentration of the filtrate at pH 12 enabled the ¹³CO₂ produced to be observed using ¹³C NMR. During incubations of H¹⁴CO₂Na with this fungus, complete conversion to other radioactive products was observed, and the significance of these results in the light of previous work is discussed.     

The effect of feeding on CO2 production and energy expenditure in ponies measured by indirect calorimetry and the 13C-bicarbonate technique

Energy expenditure (EE) can be estimated based on respiratory gas exchange measurements, traditionally done in respiration chambers by indirect calorimetry (IC). However, the ¹³C-bicarbonate technique (¹³C-BT) might be an alternative minimal invasive method for estimation of CO₂ production and EE in the field. In this study, four Shetland ponies were used to explore the effect of feeding on CO₂ production and EE measured simultaneously by IC and ¹³C-BT. The ponies were individually housed in respiration chambers and received either a single oral or intravenous (IV) bolus dose of ¹³C-labelled sodium bicarbonate (NaH¹³CO₃). The ponies were fed haylage 3 h before (T₋₃), simultaneously with (T₀) or 3 h after (T₊₃) administration of ¹³C-bicarbonate. The CO₂ produced and O₂ consumed by the ponies were measured for 6 h with both administration routes of ¹³C-bicarbonate at the three different feeding times. Feeding time affected the CO₂ production (P<0.001) and O₂ consumption (P<0.001), but not the respiratory quotient (RQ) measured by IC. The recovery factor (RF) of ¹³C in breath CO₂ was affected by feeding time (P<0.01) and three different RF were used in the calculation of CO₂ production measured by ¹³C-BT. An average RQ was used for the calculations of EE. There was no difference between IC and ¹³C-BT for estimation of CO₂ production. An effect of feeding time (P<0.001) on the estimated EE was found, with higher EE when feed was offered (T₀ and T₊₃) compared with when no feed was available (T₋₃) during measurements. In conclusion, this study showed that feeding time affects the RF and measurements of CO₂ production and EE. This should be considered when the ¹³C-BT is used in the field. IV administration of ¹³C-bicarbonate is recommended in future studies with horses to avoid complex ¹³C enrichment-time curves with maxima and shoulders as observed in several experiments with oral administration of ¹³C-bicarbonate.     

The influence of seawater carbonate ion concentration [CO32−] on the stable carbon isotope composition of the planktic foraminifera species Globorotalia inflata

We sampled the upper water column for living planktic foraminifera along the SW-African continental margin. The species Globorotalia inflata strongly dominates the foraminiferal assemblages with an overall relative abundance of 70–90%. The shell δ¹⁸O and δ¹³C values of G. inflata were measured and compared to the predicted oxygen isotope equilibrium values (δ¹⁸O_eq) and to the carbon isotope composition of the total dissolved inorganic carbon (δ¹³C_DIC) of seawater. The δ¹⁸O of G. inflata reflects the general gradient observed in the predicted δ¹⁸O_eq profile, while the δ¹³C of G. inflata shows almost no variation with depth and the reflection of the δ¹³C_DIC in the foraminiferal shell seems to be covered by other effects. We found that offsets between δ¹⁸O_shell and predicted δ¹⁸O_eq in the surface mixed layer do not correlate to changes in seawater [CO₃²⁻].To calculate an isotopic mass balance of depth integrated growth, we used the oxygen isotope composition of G. inflata to estimate the fraction of the total shell mass that is grown within each plankton tow depth interval of the upper 500 m of the water column. This approach allows us to calculate the Δδ¹³C_{interval added-DIC}; i.e. the isotopic composition of calcite that was grown within a given depth interval. Our results consistently show that the Δδ¹³C_IA-DIC correlates negatively with in situ measured [CO₃²⁻] of the ambient water. Using this approach, we found Δδ¹³C_IA-DIC/[CO₃²⁻] slopes for G. inflata in the large size fraction (250–355 μm) of − 0.013‰ to 0.015‰ (μmol kg^− 1)^− 1 and of − 0.013‰ to 0.017‰ (μmol kg^− 1)^− 1 for the smaller specimens (150–250 μm). These slopes are in the range of those found for other non-symbiotic species, such as Globigerina bulloides, from laboratory culture experiments. Since the Δδ¹³C_IA-DIC/[CO₃²⁻] slopes from our field data are nearly identical to the slopes established from laboratory culture experiments we assume that the influence of other effects, such as temperature, are negligibly small. If we correct the δ¹³C values of G. inflata for a carbonate ion effect, the δ¹³C_shell and δ¹³C_DIC are correlated with an average offset of 2.11.     

Resveratrol and para-coumarate serve as ring precursors for coenzyme Q biosynthesis

Coenzyme Q (Q or ubiquinone) is a redox-active polyisoprenylated benzoquinone lipid essential for electron and proton transport in the mitochondrial respiratory chain. The aromatic ring 4-hydroxybenzoic acid (4HB) is commonly depicted as the sole aromatic ring precursor in Q biosynthesis despite the recent finding that para-aminobenzoic acid (pABA) also serves as a ring precursor in Saccharomyces cerevisiae Q biosynthesis. In this study, we employed aromatic ¹³C₆-ring-labeled compounds including ¹³C₆-4HB, ¹³C₆-pABA, ¹³C₆-resveratrol, and ¹³C₆-coumarate to investigate the role of these small molecules as aromatic ring precursors in Q biosynthesis in Escherichia coli, S. cerevisiae, and human and mouse cells. In contrast to S. cerevisiae, neither E. coli nor the mammalian cells tested were able to form ¹³C₆-Q when cultured in the presence of ¹³C₆-pABA. However, E. coli cells treated with ¹³C₆-pABA generated ¹³C₆-ring-labeled forms of 3-octaprenyl-4-aminobenzoic acid, 2-octaprenyl-aniline, and 3-octaprenyl-2-aminophenol, suggesting UbiA, UbiD, UbiX, and UbiI are capable of using pABA or pABA-derived intermediates as substrates. E. coli, S. cerevisiae, and human and mouse cells cultured in the presence of ¹³C₆-resveratrol or ¹³C₆-coumarate were able to synthesize ¹³C₆-Q. Future evaluation of the physiological and pharmacological responses to dietary polyphenols should consider their metabolism to Q.     

Seasonal variations in bulk tissue, fatty acid and monosaccharide δC values of leaves from mesotrophic grassland plant communities under different grazing managements

Leaves of 26 grass, herb, shrub and tree species were collected from mesotrophic grasslands to assess natural variability in bulk, fatty acid and monosaccharide δ¹³C values under different grazing management (cattle- or deer-grazed) on three sample dates (May, July and October) such that interspecific and spatiotemporal variations in whole leaf tissues and compound-specific δ¹³C values could be determined. The total mean leaf bulk δ¹³C value for plants was −28.9‰ with a range of values spanning 7.5‰. Significant interspecific variation between bulk leaf δ¹³C values was only determined in October (P = <0.001) when δ¹³C values of the leaf tissues from both sites was on average 1.5‰ depleted compared to during July and May. Samples from May were significantly different between fields (P = 0.03) indicating an effect from deer- or cattle-grazing in young leaves. The average individual monosaccharide δ¹³C value was 0.8‰ higher compared with whole leaf tissues. Monosaccharides were the most abundant components of leaf biomass, i.e. arabinose, xylose, mannose, galactose and glucose, and therefore, fluctuations in their individual δ¹³C values had a major influence on bulk δ¹³C values. An average depletion of ca. 1‰ in the bulk δ¹³C values of leaves from the deer-grazed field compared to the cattle-grazed field could be explained by a general depletion of 1.1‰ in glucose δ¹³C values, as glucose constituted >50% total leaf monosaccharides. In October, δ¹³C values of all monosaccharides varied between species, with significant variation in δ¹³C values of mannose and glucose in July, and mannose in May. This provided an explanation for the noted variability in the tissue bulk δ¹³C values observed in October 1999. The fatty acids C_16:0, C_18:2 and C_18:3 were highly abundant in all plant species. Fatty acid δ¹³C values were lower than those of bulk leaf tissues; average values of −37.4‰ (C_16:0), −37.0‰ (C_18:2) and −36.5‰ (C_18:3) were determined. There was significant interspecific variation in the δ¹³C values of all individual fatty acids during October and July, but only for C_18:2 in May (P = <0.05). This indicated that seasonal trends observed in the δ¹³C values of individual fatty acids were inherited from the isotopic composition of primary photosynthate. However, although wide diversity in δ¹³C values of grassland plants ascribed to grazing management, interspecific and spatiotemporal influences was revealed, significant trends (P = <0.0001) for fatty acid and monosaccharide δ¹³C values: δ¹³C_16:0 < δ¹³C_18:2 < δ¹³C_18:3 and δ¹³C_arabinose > δ¹³C_xylose > δ¹³C_glucose > δ¹³C_galactose, respectively, previously described, appear consistent across a wide range of species at different times of the year in fields under different grazing regimes.     

Azolla-Anabaena Relationship : XIII. Fixation of [N]N(2)

The major radioactive products of the fixation of [¹³N]N₂ by Azolla caroliniana Willd.-Anabaena azollae Stras. were ammonium, glutamine, and glutamate, plus a small amount of alanine. Ammonium accounted for 70 and 32% of the total radioactivity recovered after fixation for 1 and 10 minutes, respectively. The presence of a substantial pool of [¹³N]N₂-derived ¹³NH₄⁺ after longer incubation periods was attributed to the spatial separation between the site of N₂-fixation (Anabaena) and a second, major site of assimilation (Azolla). Initially, glutamine was the most highly radioactive organic product formed from [¹³N]N₂, but after 10 minutes of fixation glutamate had 1.5 times more radiolabel than glutamine. These kinetics of radiolabeling, along with the effects of inhibitors of glutamine synthetase and glutamate synthase on assimilation of exogenous and [¹³N]N₂-derived ¹³NH₄⁺, indicate that ammonium assimilation occurred by the glutamate synthase cycle and that glutamate dehydrogenase played little or no role in the synthesis of glutamate by Azolla-Anabaena.     

Respiration of 13C-Labeled Substrates Added to Soil in the Field and Subsequent 16S rRNA Gene Analysis of 13C-Labeled Soil DNA

Our goal was to develop a field soil biodegradation assay using ¹³C-labeled compounds and identify the active microorganisms by analyzing 16S rRNA genes in soil-derived ¹³C-labeled DNA. Our biodegradation approach sought to minimize microbiological artifacts caused by physical and/or nutritional disturbance of soil associated with sampling and laboratory incubation. The new field-based assay involved the release of ¹³C-labeled compounds (glucose, phenol, caffeine, and naphthalene) to soil plots, installation of open-bottom glass chambers that covered the soil, and analysis of samples of headspace gases for ¹³CO₂ respiration by gas chromatography/mass spectrometry (GC/MS). We verified that the GC/MS procedure was capable of assessing respiration of the four substrates added (50 ppm) to 5 g of soil in sealed laboratory incubations. Next, we determined background levels of ¹³CO₂ emitted from naturally occurring soil organic matter to chambers inserted into our field soil test plots. We found that the conservative tracer, SF₆, that was injected into the headspace rapidly diffused out of the soil chamber and thus would be of little value for computing the efficiency of retaining respired ¹³CO₂. Field respiration assays using all four compounds were completed. Background respiration from soil organic matter interfered with the documentation of in situ respiration of the slowly metabolized (caffeine) and sparingly soluble (naphthalene) compounds. Nonetheless, transient peaks of ¹³CO₂ released in excess of background were found in glucose- and phenol-treated soil within 8 h. Cesium-chloride separation of ¹³C-labeled soil DNA was followed by PCR amplification and sequencing of 16S rRNA genes from microbial populations involved with ¹³C-substrate metabolism. A total of 29 full sequences revealed that active populations included relatives of Arthrobacter, Pseudomonas, Acinetobacter, Massilia, Flavobacterium, and Pedobacter spp. for glucose; Pseudomonas, Pantoea, Acinetobacter, Enterobacter, Stenotrophomonas, and Alcaligenes spp. for phenol; Pseudomonas, Acinetobacter, and Variovorax spp. for naphthalene; and Acinetobacter, Enterobacter, Stenotrophomonas, and Pantoea spp. for caffeine.     

Dynamic aspects of extracellular loop region as a proton release pathway of bacteriorhodopsin studied by relaxation time measurements by solid state NMR

Local dynamics of interhelical loops in bacteriorhodopsin (bR), the extracellular BC, DE and FG, and cytoplasmic AB and CD loops, and helix B were determined on the basis of a variety of relaxation parameters for the resolved ¹³C and ¹⁵N signals of [1-¹³C]Tyr-, [¹⁵N]Pro- and [1-¹³C]Val-, [¹⁵N]Pro-labeled bR. Rotational echo double resonance (REDOR) filter experiments were used to assign [1-¹³C]Val-, [¹⁵N]Pro signals to the specific residues in bR. The previous assignments of [1-¹³C]Val-labeled peaks, 172.9 or 171.1 ppm, to Val69 were revised: the assignment of peak, 172.1 ppm, to Val69 was made in view of the additional information of conformation-dependent ¹⁵N chemical shifts of Pro bonded to Val in the presence of ¹³C-¹⁵N correlation, although no assignment of peak is feasible for ¹³C nuclei not bonded to Pro. ¹³C or ¹⁵N spin-lattice relaxation times (T₁), spin-spin relaxation times under the condition of CP-MAS (T₂), and cross relaxation times (T_CH and T_NH) for ¹³C and ¹⁵N nuclei and carbon or nitrogen-resolved, ¹H spin-lattice relaxation times in the rotating flame (¹H T_1ρ) for the assigned signals were measured in [1-¹³C]Val-, [¹⁵N]Pro-bR. It turned out that V69-P70 in the BC loop in the extracellular side has a rigid β-sheet in spite of longer loop and possesses large amplitude motions as revealed from ¹³C and ¹⁵N conformation-dependent chemical shifts and T₁, T₂, ¹H T_1ρ and cross relaxation times. In addition, breakage of the β-sheet structure in the BC loop was seen in bacterio-opsin (bO) in the absence of retinal.     

Carbon-13 nuclear magnetic resonance studies of polyamino acids: the helix-coil transition of poly-L-lysine

The helix-coil transition of poly-l-lysine hydrochloride ((Lys)_n) in aqueous solution has been studied by ¹³C Fourier-transform nuclear magnetic resonance spectroscopy. As reference compounds dodeca-l-lysine hydrobromide ((Lys)^?₁₂, tri-l-lysine hydrochloride ((Lys)₃), and l-lysine hydrochloride (Lys), have been also studied by the same method. It is found that ¹³C spin-lattice relaxation times t₁ of the carbonyl and the side-chain carbons decrease sharply at pD 10.2 which is the midpoint of the transition from the random-coil to the α-helix. Similarly the T₁ values of the carbonyl groups of (Lys)^?₁₂ decrease at this point in a more moderate way, while no change is observed for those of the side-chain carbons. This is interpreted in terms of the reduced α-helicity involved for (Lys)^?₁₂.The variation of ¹³C chemical shifts with pD for (Lys)_n and (Lys)^?₁₂ show the same trend:downfield shifts at higher pD. Furthermore, nonterminal and C-terminal residues of (Lys)₃ show similar behavior. Thus it is concluded that the ¹³C chemical shift changes are caused mainly by the pD changes and not by the conformational transition. Conversion from α-helix to β-structure by elevation of temperature at pD 11.2 results in narrowing and downfield shifts of the ¹³C resonances of (Lys)_n.     

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.     

Biosynthesis of the iridoid glucoside,lamalbid, in Lamium barbatum

The biosynthesis of the iridoid glucoside lamalbid in Lamium barbatum, a plant species in the Lamiaceae, was investigated by administrating ¹³C-labeled intermediates of MVA and MEP pathways, respectively. The results demonstrated that [3,4,5-¹³C₃]1-deoxy-d-xylulose 5-phosphate could be incorporated into lamalbid, whereas the incorporation of [2-¹³C₁]mevalonolactone was not observed. Based on the ¹³C labeling pattern of lamalbid and the incorporation data, we deduce that the iridoid glucoside in L. barbatum is biosynthesized through the MEP pathway, whereas the classic MVA pathway is not utilized.     

Stable Isotope Labeled n-Alkanes to Assess Digesta Passage Kinetics through the Digestive Tract of Ruminants

We describe the use of carbon stable isotope (¹³C) labeled n-alkanes as a potential internal tracer to assess passage kinetics of ingested nutrients in ruminants. Plant cuticular n-alkanes originating from intrinsically ¹³C labeled ryegrass plants were pulse dosed intraruminally in four rumen-cannulated lactating dairy cows receiving four contrasting ryegrass silage treatments that differed in nitrogen fertilization level (45 or 90 kg nitrogen ha⁻¹) and maturity (early or late). Passage kinetics through the gastrointestinal tract were derived from the δ¹³C (i.e. the ratio ¹³C:¹²C) in apparently undigested fecal material. Isotopic enrichment was observed in a wide range of long-chain n-alkanes (C₂₇–C₃₆) and passage kinetics were determined for the most abundant C₂₉, C₃₁ and C₃₃ n-alkanes, for which a sufficiently high response signal was detected by combustion isotope ratio mass spectrometry. Basal diet treatment and carbon chain length of n-alkanes did not affect fractional passage rates from the rumen (K ₁) among individual n-alkanes (3.71–3.95%/h). Peak concentration time and transit time showed a quantitatively small, significant (p≤0.002) increase with carbon chain length. K ₁ estimates were comparable to those of the ¹³C labeled digestible dry matter fraction (3.38%/h; r = 0.61 to 0.71; p≤0.012). A literature review has shown that n-alkanes are not fermented by microorganisms in the rumen and affirms no preferential depletion of ¹³C versus ¹²C. Our results suggest that ¹³C labeled n-alkanes can be used as nutrient passage tracers and support the reliability of the δ¹³C signature of digestible feed nutrients as a tool to measure nutrient-specific passage kinetics.     

para-Aminobenzoic Acid Is a Precursor in Coenzyme Q6 Biosynthesis in Saccharomyces cerevisiae

Coenzyme Q (ubiquinone or Q) is a crucial mitochondrial lipid required for respiratory electron transport in eukaryotes. 4-Hydroxybenozoate (4HB) is an aromatic ring precursor that forms the benzoquinone ring of Q and is used extensively to examine Q biosynthesis. However, the direct precursor compounds and enzymatic steps for synthesis of 4HB in yeast are unknown. Here we show that para-aminobenzoic acid (pABA), a well known precursor of folate, also functions as a precursor for Q biosynthesis. A hexaprenylated form of pABA (prenyl-pABA) is normally present in wild-type yeast crude lipid extracts but is absent in yeast abz1 mutants starved for pABA. A stable ¹³C₆-isotope of pABA (p- amino[aromatic-¹³C₆]benzoic acid ([¹³C₆]pABA)), is prenylated in either wild-type or abz1 mutant yeast to form prenyl-[¹³C₆]pABA. We demonstrate by HPLC and mass spectrometry that yeast incubated with either [¹³C₆]pABA or [¹³C₆]4HB generate both ¹³C₆-demethoxy-Q (DMQ), a late stage Q biosynthetic intermediate, as well as the final product ¹³C₆-coenzyme Q. Pulse-labeling analyses show that formation of prenyl-pABA occurs within minutes and precedes the synthesis of Q. Yeast utilizing pABA as a ring precursor produce another nitrogen containing intermediate, 4-imino-DMQ₆. This intermediate is produced in small quantities in wild-type yeast cultured in standard media and in abz1 mutants supplemented with pABA. We suggest a mechanism where Schiff base-mediated deimination forms DMQ₆ quinone, thereby eliminating the nitrogen contributed by pABA. This scheme results in the convergence of the 4HB and pABA pathways in eukaryotic Q biosynthesis and has implications regarding the action of pABA-based antifolates.     

Passage kinetics of concentrates in dairy cows measured with carbon stable isotopes

Fractional passage rates form a fundamental element within modern feed evaluation systems for ruminants, but knowledge on feed-specific fractional passage is largely lacking. Commonly applied tracer techniques based on externally applied markers, such as chromium-mordanted neutral detergent fibre (Cr-NDF), have been criticised for behaving differently to feed particles. This study describes the use of the carbon stable isotope ratio (¹³C : ¹²C) as an internal digesta marker to quantify the fractional passage rate of concentrates through the digestive tract of dairy cows. In a crossover study, five dairy cows were fed low (24.6%) and high (52.6%) levels of concentrates (dry matter (DM) basis) and received a pulse-dosed Cr-NDF and ¹³C isotopes. The latter was administered orally by exchanging part of the dietary concentrates of low ¹³C natural abundance with a pulse dose of maize bran-based concentrates of high ¹³C natural abundance. Fractional passage rates from the rumen (K₁) and from the large intestine (K₂) were determined from faecal marker concentrations of Cr-NDF and of ¹³C in the DM (¹³C-DM), NDF (¹³C-NDF) and neutral detergent soluble (¹³C-NDS). No differences in K₁ estimates were found for the two concentrate levels fed but significant differences between markers (P<0.001) were observed. Faecal Cr-NDF excretions gave lower K₁ estimates (0.037–0.039/h) than ¹³C-DM (0.054–0.056/h) and ¹³C-NDF (0.061–0.063/h). The ¹³C-NDS was calculated by the difference of ¹³C in the DM and NDF, and K₁ values (0.039–0.043/h) were comparable to Cr-NDF. Total mean retention time was considerably higher for Cr-NDF (40.9–42.0 h) as compared to ¹³C-DM and ¹³C-NDF (32.0–33.5 h; P<0.001). The accuracy of the curve fits for Cr-NDF and ¹³C-DM and ¹³C-NDF was overall good (mean prediction error of 9.9–13.9%). Fractional passage rate of Cr-NDF was comparable to studies where this marker was assumed to represent the fractional passage of roughages. However, K₁ estimates based on the ¹³C : ¹²C ratio varied considerably from studies based on external markers. Our results suggest that the use of ¹³C isotopes as digesta passage markers can provide feed component-specific K₁ estimates for concentrates and provides new insight into passage kinetics of NDF from technologically treated compound feed.     

In vivo enzymology: 13C NMR measurement of a kinetic isotope effect for methanol oxidation in Methylosinus trichosporium OB3b

The competitive oxidation of ¹³CH₃OH and ¹³CD₃OH has been observed using in vivo ¹³C NMR spectroscopy. Simultaneous ¹H and ²H decoupling gave isotopically shifted ¹³C singlets for the two methanol isotopomers. The measured enzymic isotope effect, kH/kD is approx. 1.8, indicating that CH bond cleavage is not rate-determining.     

13C isotope fractionation during rhizosphere respiration of C3 and C4 plants

Stable carbon isotopes are used extensively to partition total soil CO₂ efflux into root-derived rhizosphere respiration or autotrophic respiration and soil-derived heterotrophic respiration. However, it remains unclear whether CO₂ from rhizosphere respiration has the same δ¹³C value as root biomass. Here we investigated the magnitude of ¹³C isotope fractionation during rhizosphere respiration relative to root biomass in six plant species. Plants were grown in a carbon-free sand-perlite medium inoculated with microorganisms from a farm soil for 62 days inside a greenhouse. We measured the δ¹³C value of rhizosphere respiration using a closed-circulation 48-hour CO₂ trapping method during 40~42 and 60~62 days after sowing. We found a consistent depletion in ¹³C (0.9~1.7‰) of CO₂ from rhizosphere respiration relative to root biomass in three C₃ species (Glycine max L. Merr., Helianthus annuus L. and Triticum aestivum L.), but a relatively large depletion in ¹³C (3.7~7.0‰) in three C₄ species (Amaranthus tricolor L., Sorghum bicolor (L.) Moench and Zea mays L. ssp. mays). Overall, our results indicate that CO₂ from rhizosphere respiration is more ¹³C-depleted than root biomass. Therefore, accounting for this ¹³C fractionation is required for accurately partitioning total soil CO₂ efflux into root-derived and soil-derived components using natural abundance stable carbon isotope methods.     

Transportation of de novo synthesized jasmonoyl isoleucine in tomato

In plants, jasmonic acid (JA) and its derivatives are thought to be involved in mobile forms of defense against biotic and abiotic stresses. In this study, the distal transport of JA-isoleucine (JA-Ile) that is synthesized de novo in response to leaf wounding in tomato (Solanum lycopersicum) plants was investigated. JA-[¹³C₆]Ile was recovered in distal untreated leaves after wounded leaves were treated with [¹³C₆]Ile. However, as [¹³C₆]Ile was also recovered in the distal untreated leaves, whether JA-Ile was synthesized in the wounded or in the untreated leaves was unclear. Hence, stem exudates were analyzed to obtain more detailed information. When [¹³C₆]Ile and [²H₆]JA were applied separately into the wounds on two different leaves, JA-[¹³C₆]Ile and [²H₆]JA-Ile were detected in the stem exudates but [²H₆]JA–[¹³C₆]Ile was not, indicating that JA was conjugated with Ile in the wounded leaf and that the resulting JA-Ile was then transported into systemic tissues. The [²H₃]JA-Ile that was applied exogenously to the wounded tissues reached distal untreated leaves within 10 min. Additionally, applying [²H₃]JA-Ile to the wounded leaves at concentrations of 10 and 60 nmol/two leaves induced the accumulation of PIN II, LAP A, and JAZ3 mRNA in the distal untreated leaves of the spr2 mutant S. lycopersicum plants. These results demonstrate the transportation of de novo synthesized JA-Ile and suggest that JA-Ile may be a mobile signal.     

Physiological characterization of recombinant inbred lines of barley with contrasting levels of carbon isotope discrimination

Background and Aims

Carbon isotope discrimination (Δ¹³C) in C₃ plants used as an indirect measure of water-use efficiency (WUE) provides a tool for selecting crops with high WUE under dry environments.

Methods

We evaluated the physiology and Δ¹³C of a set of 8 F₅ recombinant inbred lines (RILs) with contrasting levels of leaf Δ¹³C derived from two parents, ‘W89001002003’ (low Δ¹³C) and ‘I60049’ (high Δ¹³C) of six-row barley (Hordeum vulgare L.) in a greenhouse and under field conditions in three locations (Lacombe, Vegreville and Castor). In the greenhouse experiment, seven days of water deficit was imposed at the stem elongation stage followed by re-watering to pre-deficit level.

Results

A significant negative relationship between WUE and leaf Δ¹³C was observed. Under water-deficit conditions, both photosynthetic rate (A) and stomatal conductance (g _s ) were significantly reduced with a strong positive correlation (r = 0.89) between the two, and the variation in g _s was proportionally greater than A. The low leaf-Δ¹³C RIL ‘147’ maintained the highest A and g _s among ten genotypes (RILs and parents) under water-deficit conditions. Leaf Δ¹³C was positively correlated with biomass and grain yield in the field trials. Multivariate analysis of leaf Δ¹³C, harvest index and plant height discriminated genotypes into three clusters: drought sensitive, drought tolerant and an intermediate type.

Conclusions

The study suggests that it is possible to select low Δ¹³C lines such as RIL ‘147’, which is able to maintain or produce high yields under low moisture conditions on the Canadian Prairies     

The yeast cloning vector YEp13 contains a tRNA3Leu gene that can mutate to an amber suppressor

We have shown that the yeast-Escherichia coli shuttle vector YEp 13 contains, as part of its yeast chromosomal segment, a tRNA₃^Leu gene. We have also isolated and characterized a variant of YEp13, namely YEp13-a, which is capable of suppressing a variety of yeast amber-suppressible alleles in vivo. YEp13-a differs from YEp13 by a single point mutation, which changes the three-nucleotide, plus-strand sequence corresponding to the tRNA₃^Leu anticodon from the normal C-A-A to C-T-A. This nucleotide change creates a site for the restriction enzyme XbaI in the suppressor tRNA₃^Leu gene. We have taken advantage of the correlation between the suppressor mutation and the XbaI site formation, to show that the tRNA₃^Leu gene on YEp13 corresponds to the genetically characterized yeast chromosomal amber suppressor SUP53. We have also shown that SUP53 is located just centromere-distal to LEU2 on chromosome III. Finally, comparison of the DNA sequence of SUP53 and its flanking regions with the sequences of other cloned yeast tRNA₃^Leu genes has revealed considerable sequence homology in the immediate 5′-flanking regions of these genes.