Solid phase (9-fluorenylmethyloxycarbonyl) synthesis and characterization of [[1-13C]Phe11]gramicidin B1

The solid phase synthesis of [[1-¹³C]Phe¹¹]Gramicidin B was successfully achieved using the 9-Fluorenylmethyloxycarbonyl protecting group. There was at least a 30% drop in yield during the adition of the three valines in positions 8, 7 and 6, which is likely due to steric hindrance caused by the steric constraints of the valine side chains. Still, the overall yield of the peptide was comparable with that obtained using thetert-butyloxycarbonyl group for protection. The synthetic Gramicidin B was completely characterized by high-pressure liquid chromatography, circular dichroism, and¹³C nuclear magnetic resonance spectra. Also reported are the single-channel conductance properties, which compare favorably with those reported earlier and demonstrate the interesting multiplicity of conductance states to be distinquishable from that of Gramicidin A. Thus, these several useful characterizations have been carried out on the same preparation.Abbreviations: IUPAC-IUB Commission recommendations are used in most cases: AcOH, acetic acid; Boc,tert-butyloxycarbonyl; CD, circular dichroism; CMR,¹³C nuclear magnetic resonance; DCC,N,N'-dicyclohexylcarbodiimide; EtOAc, ethyl acetate; Fmoc, 9-fluorenylmethyloxycarbonyl; HOBt, 1-hydroxybenzot iazole; HPLC, high-pressure liquid chromatography; MeOH, methanol;-OSu,N-hydroxysuccinimide ester; TFA, trifluoroacetic acid; t.l.c., thin-layer chromatography; Me₂SO-d ₆, dimethyl-d ₆-sulfoxide; CMA, chloroform-methanol-acetic acid; DMF, dimethyl for-mamide; DIEA, diisoproprylethylamine.     

The dimeric nature of the gramicidin A transmembrane channel: Conductance and fluorescence energy transfer studies of hybrid channels

Gramicidin A, a linear peptide antibiotic, makes membranes permeable to alkali cations and hydrogen ions by forming transmembrane channels. We report here conductance and fluorescence energy transfer studies of channels containing two kinds of gramicidin. These studies of hybrid channels were designed to determine the number of molecules in a channel. The gramicidins studied were gramicidin A, dansyl gramicidin C, the p-phenylazobenzene sulfonyl derivative of gramicidin C (PABS⁴ gramicidin C), and the 4-(diethylamino)-phenylazobenzene-4-sulfonyl chloride derivative of gramicidin C (DPBS gramicidin C). The dansyl, PABS and DPBS groups were linked to the hydroxyl group of tyrosine 11 in gramicidin C. The single-channel conductance of PABS gramicidin C in planar bilayer membranes is 0.68 that of gramicidin A. Membranes containing both PABS gramicidin C and gramicidin A exhibit three kinds of channels: a pure gramicidin A, a pure PABS gramicidin C channel, and a hybrid channel with an intermediate conductance (0.82 that of gramicidin A). The dependence of the frequencies of these three kinds of channels on the mole fractions of gramicidin A and PABS gramicidin C in the membrane-forming solution fits a dimer model. Fluorescence energy transfer was used as a complementary means of ascertaining the frequency of hybrid channels. Dansyl gramicidin C was the fluorescent energy donor and DPBS gramicidin C was the energy acceptor. The efficiency of energy transfer between these chromophores in hybrid channels in liposomes was 75%. The relative quantum yield of the dansyl fluorescence was measured as a function of the mole fraction of DPBS gramicidin C. These fluorescence studies, like the single-channel conductance measurements, showed that there are two molecules of gramicidin in a channel. The study of hybrid species by conductance and fluorescence techniques should be generally useful in elucidating the subunit structure of oligomeric assemblies in membranes.     

Fermentation and Cost-Effective 13C/15N Labeling of the Nonribosomal Peptide Gramicidin S for Nuclear Magnetic Resonance Structure Analysis

Gramicidin S (GS) is a nonribosomally synthesized decapeptide from Aneurinibacillus migulanus. Its pronounced antibiotic activity is attributed to amphiphilic structure and enables GS interaction with bacterial membranes. Despite its medical use for over 70 years, the peptide-lipid interactions of GS and its molecular mechanism of action are still not fully understood. Therefore, a comprehensive structural analysis of isotope-labeled GS needs to be performed in its biologically relevant membrane-bound state, using advanced solid-state nuclear magnetic resonance (NMR) spectroscopy. Here, we describe an efficient method for producing the uniformly ¹³C/¹⁵N-labeled peptide in a minimal medium supplemented by selected amino acids. As GS is an intracellular product of A. migulanus, we characterized the producer strain DSM 5759 (rough-convex phenotype) and examined its biosynthetic activity in terms of absolute and biomass-dependent peptide accumulation. We found that the addition of either arginine or ornithine increases the yield only at very high supplementing concentrations (1% and 0.4%, respectively) of these expensive ¹³C/¹⁵N-labeled amino acids. The most cost-effective production of ¹³C/¹⁵N-GS, giving up to 90 mg per gram of dry cell weight, was achieved in a minimal medium containing 1% ¹³C-glycerol and 0.5% ¹⁵N-ammonium sulfate, supplemented with only 0.025% of ¹³C/¹⁵N-phenylalanine. The 100% efficiency of labeling is corroborated by mass spectrometry and preliminary solid-state NMR structure analysis of the labeled peptide in the membrane-bound state.     

Probing Early Tumor Response to Radiation Therapy Using Hyperpolarized [1-13C]pyruvate in MDA-MB-231 Xenografts

Following radiation therapy (RT), tumor morphology may remain unchanged for days and sometimes weeks, rendering anatomical imaging methods inadequate for early detection of therapeutic response. Changes in the hyperpolarized [1-¹³C]lactate signals observed in vivo following injection of pre-polarized [1-¹³C]pyruvate has recently been shown to be a marker for tumor progression or early treatment response. In this study, the feasibility of using ¹³C metabolic imaging with [1-¹³C]pyruvate to detect early radiation treatment response in a breast cancer xenograft model was demonstrated in vivo and in vitro. Significant decreases in hyperpolarized [1-¹³C]lactate relative to [1-¹³C]pyruvate were observed in MDA-MB-231 tumors 96 hrs following a single dose of ionizing radiation. Histopathologic data from the treated tumors showed higher cellular apoptosis and senescence; and changes in the expression of membrane monocarboxylate transporters and lactate dehydrogenase B were also observed. Hyperpolarized ¹³C metabolic imaging may be a promising new tool to develop novel and adaptive therapeutic regimens for patients undergoing RT.     

Gas Exchange, Stomatal Behavior, and deltaC Values of the flacca Tomato Mutant in Relation to Abscisic Acid

The relationship between stomatal conductance and capacity for assimilation was investigated in flacca, a mutant of tomato (Lycopersicon esculentum Mill.) that has abnormal stomatal behavior and low abscisic acid (ABA) content. The assimilation capacity, determined by measuring assimilation rate as a function of intercellular CO₂ pressure, did not differ in leaves of flacca and its parent variety, Rheinlands Ruhm (RR). On the other hand, stomatal conductance of flacca leaves was greater than that of RR, and could be phenotypically reverted by spraying with 30 micromolar ABA. Stomatal conductance of flacca leaves was also reduced by increasing CO₂ pressure, increasing leaf to air vapor pressure difference, and decreasing quantum flux, irrespective of ABA treatment.

The high conductance of flacca leaves resulted in a high intercellular CO₂ pressure. This allowed greater discrimination against ¹³CO₂, as evidenced by more negative δ ¹³C values for flacca as compared to RR. The δ ¹³C values of both flacca and RR plants as influenced by ABA treatment were consistent with predictions based on gas exchange measurements, using a recent model of discrimination.

     

Tetrapyrrole biosynthesis in Anacystis nidulans; incorporation of [1-13C]-, [2-13C]-, [1,2-13C]- and [2-13C, 2-2H3]acetate

Labelling experiments with [2-¹³C]- and [1,2-¹³C]acetate showed that both photopigments of Anacystis nidulans, chlorophyll a and phycocyanobilin, share a common biosynthetic pathway from glutamate. The fate of deuterium during these biosynthetic events was studied using [2-¹³C, 2-²H₃]acetate as a precursor and determining the labelling pattern by ¹³C NMR spectroscopy with simultaneous [¹H, ²H]-broadband decoupling. The loss of ²H (ca 20%) from the precursor occurred at an early stage during the tricarboxylic acid cycle. After formation of glutamate there was no further loss of ²H in the assembly of the cyclic tetrapyrrole intermediates or during decarboxylation and modification of the side-chains. Thus the labelling data support a divergence in the pathway to cyclic and linear tetrapyrroles after protoporphyrin IX.     

Synthesis,in vitro and in vivo evaluation of [11C]MMTP: A potential PET ligand for mGluR1 receptors

Synthesis, in vitro and in vivo evaluation of [O-methyl-¹¹C]dimethylamino-3(4-methoxyphenyl)-3H-pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin-4-one (1), a potential imaging agent for mGluR1 receptors using PET are described. Synthesis of the corresponding desmethyl precursor 2 was achieved by demethylation of the methoxyphenyl compound 1 in 90% yield. Methylation using [¹¹C]MeOTf in presence of NaOH afforded [¹¹C]1 in 30% yield (EOS) with >99% chemical and radiochemical purities and with a specific activity of 3–5 Ci/μmol (n = 6). The total synthesis time was 30 min from EOB. The radiotracer selectively labeled mGluR1 receptors in slide-mounted sections of postmortem human brain containing cerebellum, hippocampus, prefrontal cortex and striatum as demonstrated by in vitro autoradiography using phosphor-imaging. PET studies in anesthetized baboon show that [¹¹C]1 penetrates the BBB and accumulates in cerebellum, a region reported to have higher expression of mGluR1. These findings suggest [¹¹C]1 is a promising PET radiotracer candidate for mGluR1.     

In vivo enzymatic activity of acetylCoA synthetase in skeletal muscle revealed by C turnover from hyperpolarized [1-C]acetate to [1-C]acetylcarnitine

Background

Acetate metabolism in skeletal muscle is regulated by acetylCoA synthetase (ACS). The main function of ACS is to provide cells with acetylCoA, a key molecule for numerous metabolic pathways including fatty acid and cholesterol synthesis and the Krebs cycle.

Methods

Hyperpolarized [1-¹³C]acetate prepared via dissolution dynamic nuclear polarization was injected intravenously at different concentrations into rats. The ¹³C magnetic resonance signals of [1-¹³C]acetate and [1-¹³C]acetylcarnitine were recorded in vivo for 1 min. The kinetic rate constants related to the transformation of acetate into acetylcarnitine were deduced from the 3 s time resolution measurements using two approaches, either mathematical modeling or relative metabolite ratios.

Results

Although separated by two biochemical transformations, a kinetic analysis of the ¹³C label flow from [1-¹³C]acetate to [1-¹³C]acetylcarnitine led to a unique determination of the activity of ACS. The in vivo Michaelis constants for ACS were K_M = 0.35 ± 0.13 mM and V_max = 0.199 ± 0.031 μmol/g/min.

Conclusions

The conversion rates from hyperpolarized acetate into acetylcarnitine were quantified in vivo and, although separated by two enzymatic reactions, these rates uniquely defined the activity of ACS. The conversion rates associated with ACS were obtained using two analytical approaches, both methods yielding similar results.

General significance

This study demonstrates the feasibility of directly measuring ACS activity in vivo and, since the activity of ACS can be affected by various pathological states such as cancer or diabetes, the proposed method could be used to non-invasively probe metabolic signatures of ACS in diseased tissue.     

Synthesis and in vitro evaluation of new derivatives of 2-substituted-6-fluorobenzo[d]thiazoles as cholinesterase inhibitors

A series of novel cholinesterase inhibitors based on 2-substituted 6-fluorobenzo[d]thiazole were synthesised and characterised by IR, ¹H, ¹³C and ¹⁹F NMR spectroscopy and HRMS. Purity was checked by elemental analyses. The novel carbamates were tested for their ability to inhibit acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The toxicity of the most active compounds was investigated using a standard in vitro test with HepG2 cells, and the ratio between biological activity and toxicity was determined. In addition, the toxicity of the most active compounds was evaluated against MCF7 cells using the xCELLigence system. Structure–activity relationships reflecting the dependence of cholinesterase inhibitors on the lipophilicity of the compounds as well as on the Taft polar and steric substituent constants are discussed. The specific orientation of the inhibitors in the binding site of acetylcholinesterase was determined using molecular docking of the most active compound.     

Buffering Capacity and Membrane H+ Conductance of Neutrophilic and Alkalophilic Gram-Positive Bacteria

Buffering capacity and membrane H⁺ conductance were examined in three gram-positive bacteria, Staphylococcus aureus, Bacillus subtilis, and Bacillus alcalophilus. An acid pulse technique was used to measure both parameters. The buffering capacity and membrane H⁺ conductance of B. alcalophilus are influenced by the pH of the medium and the culture conditions. Suspensions of B. alcalophilus cells from both H. A. medium and l-malate medium cultures grown at pH 10.5 exhibited higher values for these parameters than cells grown at pH 8.5. B. alcalophilus grown aerobically had a lower buffering capacity and a lower membrane conductance for protons than the neutrophilic bacteria S. aureus and B. subtilis. Fermenting cells exhibited significantly higher values for both variables than respiring cells.Most microorganisms are neutrophiles, since they survive only at pH values ranging from 5 to 8.5 and exhibit maximum growth rates at pH 7.4 (24). There is, however, a diverse group of bacteria that thrive in highly alkaline environments (11). Bacillus alcalophilus is an obligate alkalophile that can grow at pH values ranging from 8.5 to 11.5, and optimum growth occurs at pH 10.6 (12). It has been suggested that the obligate alkalophiles fail to grow at neutral pH because their membranes become leaky (2). In addition, Krulwich et al. (13) encountered difficulties when they measured the buffering capacities (as determined with suspensions of cells permeabilized with Triton X-100 or n-butanol) of two alkalophilic bacteria, B. alcalophilus and Bacillus firmus RAB, at pH values below 6.5 due to loss of cell integrity.The work presented here is the last part of an extensive study of the buffering capacity and membrane H⁺ conductance of gram-negative and gram-positive bacteria (17–22). We used a method in which the decay of an acid pulse is used to determine both parameters (15). By using this approach, we avoided the technical problems of the method involving permeabilizing cells, as described by Krulwich et al. (13). Here we report buffering capacity and membrane H⁺ conductance values for the following gram-positive bacteria: two mesophilic neutrophiles, Staphylococcus aureus and Bacillus subtilis, and the obligately alkalophilic bacillus B. alcalophilus. We measured both parameters in B. alcalophilus cells grown in two media at pH 8.5 and 10.5.     

Aspergillus oryzae type III polyketide synthase CsyB uses a fatty acyl starter for the biosynthesis of csypyrone B compounds

Csypyrones B1, B2 and B3 are α-pyrones that can be obtained from Aspergillus oryzae expressing CsyB, which is a type III polyketide synthase. We investigated the biosynthesis of the csypyrone B compounds using [1-¹³C] and [2-¹³C] acetate feeding experiments. ¹³C NMR analyses of the methyl esters of the csypyrone B compounds fed with the ¹³C-labeled acetates showed that the carboxyl carbons of the csypyrone B side-chains were derived from the C-2 methyl carbon of the acetate. These results indicated that fatty acyl starters are involved in the CsyB reaction and that the csypyrone B compounds are formed by the oxidation of side-chains by the host fungus.     

Enzymatic preparation of [1, 3-13C]dihydroxyacetone phosphate from [13C]methanol and hydroxypyruvate using the methanol-assimilating system of methylotrophic yeasts

Dihydroxyacetoone synthase (EC 2.2.1.3), which is a key enzyme of the C₁-compound-assimilating pathway in yeasts, catalyzes transketolation between formaldehyde and hydroxypyruvate, leading to the formation of dihydroxyacetone and CO₂. When [¹³C]formaldehyde was used as a substrate with dihydroxyacytone synthase from Candida boidinii 2201, ¹³C was confirmed to be incorporated to the C-1 and C-3 positions of dihydroxyacetone, and the ¹³C content of each carbon (atoms/100 atoms) was estimated to be 50%. [¹³C]Methanol was also useful for the enrichment of dihydroxyacetone with ¹³C, when alcohol oxidase from a methylotrophic yeast was added for the conversion of methanol to formaldehyde. A fed-batch reaction with periodic addition of the substrates was required for the accumalation of ¹³C-labelled dihydroxyacetone at a higher concentration, because the enzyme system was relatively susceptible to the C donor, formaldehyde or methanol. The optimum conditions for the production gave 160mM (14.4 mg/ml) dihydroxyacetone for 180 min; the molar yield relative to methanol added was 80%. Diyhdroxyacetone kinase (EC 2.7.1.29) from methanol-grown Hansenula polymorpha CBS 4732 was a suitable enzyme for the phosphorylation of dihydroxyacytone. The phosphorylation system, comprising of dihydroxyacetone kinase, adenylate kinase, and ATP, could be coupled with the system for dihydroxyacetone production. A fed-batch reaction afforded 185 mM [1, 3-¹³C]dihydroxyacetone phosphate from [¹³C]methanol; the molar yield of the ester relative to methanol added was 92.5%     

Significance of mesophyll conductance for photosynthetic capacity and water-use efficiency in response to alkaline stress in Populus cathayana seedlings

Cuttings of Populus cathayana were exposed to three different alkaline regimes (0, 75, and 150 mM Na₂CO₃) in a semicontrolled environment. The net photosynthesis rate (P _N), mesophyll conductance (g _m), the relative limitations posed by stomatal conductance (L _s) and by mesophyll conductance (L _m), photosynthetic nitrogen-use efficiency (PNUE), carbon isotope composition (δ¹³C), as well as specific leaf area (SLA) were measured. P _N decreased due to alkaline stress by an average of 25% and g _m decreased by an average of 57%. Alkaline stress caused an increase of L _m but not L _s, with average L _s of 26%, and L _m average of 38% under stress conditions. Our results suggested reduced assimilation rate under alkaline stress through decreased mesophyll conductance in P. cathayana. Moreover, alkaline stress increased significantly δ¹³C and it drew down CO₂ concentration from the substomatal cavities to the sites of carboxylation (C _i-C _c), but decreased PNUE. Furthermore, a relationship was found between PNUE and C _i-C _c. Meanwhile, no correlation was found between δ¹³C and C _i/C _a, but a strong correlation was proved between δ¹³C and C _c/C _a, indicating that mesophyll conductance was also influencing the ¹³C/¹²C ratio of leaf under alkaline stress.     

Gramicidin Channels Are Internally Gated

Gramicidin channels are archetypal molecular subjects for solid-state NMR studies and investigations of single-channel or cation conductance. Until now, the transitions between on and off conductance states have been thought, based on multichannel studies, to represent monomer ↔ dimer reactions. Here we use a single-molecule deposition method (vesicle fusion to a planar bilayer) to show that gramicidin dimer channels do not normally dissociate when conductance terminates. Furthermore, the observation of two ¹³C peaks in solid-state NMR indicates very stable dichotomous conformations for both the first and second peptide bonds in the monomers, and a two-dimensional chemical exchange spectrum with a 12-s mixing time demonstrates that the Val₁ carbonyl conformations exchange slowly, with lifetimes of several seconds. It is proposed that gramicidin channels are gated by small conformational changes in the channel near the permeation pathway. These studies demonstrate how regulation of conformations governing closed ↔ open transitions may be achieved and studied at the molecular level.     

Tetraphyllin B from Adenia digitata

Tetraphyllin B has been isolated in good yield from the South African plant Adenia digitata. Its structure was established by ¹H and ¹³C NMR.     

The oxidative TCA cycle operates during methanotrophic growth of the Type I methanotroph Methylomicrobium buryatense 5GB1

Methanotrophs are a group of bacteria that use methane as sole carbon and energy source. Type I methanotrophs are gamma-proteobacterial methanotrophs using the ribulose monophosphate cycle (RuMP) cycle for methane assimilation. In order to facilitate metabolic engineering in the industrially promising Type I methanotroph Methylomicrobium buryatense 5GB1, flux analysis of cellular metabolism is needed and ¹³C tracer analysis is a foundational tool for such work. This biological system has a single-carbon input and a special network topology that together pose challenges to the current well-established methodology for ¹³C tracer analysis using a multi-carbon input such as glucose, and to date, no ¹³C tracer analysis of flux in a Type I methanotroph has been reported. In this study, we showed that by monitoring labeling patterns of several key intermediate metabolites in core metabolism, it is possible to quantitate the relative flux ratios for important branch points, such as the malate node. In addition, it is possible to assess the operation of the TCA cycle, which has been thought to be incomplete in Type I methanotrophs. Surprisingly, our analysis provides direct evidence of a complete, oxidative TCA cycle operating in M. buryatense 5GB1 using methane as sole carbon and energy substrate, contributing about 45% of the total flux for de novo malate production. Combined with mutant analysis, this method was able to identify fumA (METBUDRAFT_1453/MBURv2__60244) as the primary fumarase involved in the oxidative TCA cycle, among 2 predicted fumarases, supported by ¹³C tracer analysis on both fumA and fumC single knockouts. Interrupting the oxidative TCA cycle leads to a severe growth defect, suggesting that the oxidative TCA cycle functions to not only provide precursors for de novo biomass synthesis, but also to provide reducing power to the system. This information provides new opportunities for metabolic engineering of M. buryatense for the production of industrially relevant products.     

Genetic Analysis of Carbon Isotope Discrimination and its Relation to Yield in a Wheat Doubled Haploid Population

Carbon isotope discrimination (Δ¹³C) is considered a useful indicator for indirect selection of grain yield (GY) in cereals. Therefore, it is important to evaluate the genetic variation in Δ¹³C and its relationship with GY. A doubled haploid (DH) population derived from a cross of two common wheat varieties, Hanxuan 10 (H10) and Lumai 14 (L14), was phenotyped for Δ¹³C in the flag leaf, GY and yield associated traits in two trials contrasted by water availability, specifically, rain‐fed and irrigated. Quantitative trait loci (QTLs) were identified by single locus and two locus QTL analyses. QTLs for Δ¹³C were located on chromosomes 1A, 2B, 3B, 5A, 7A and 7B, and QTLs for other traits on all chromosomes except 1A, 4D, 5A, 5B and 6D. The population selected for high Δ¹³C had an increased frequency of QTL for high Δ¹³C, GY and number of spikes per plant (NSP) when grown under rain‐fed conditions and only for high Δ¹³C and NSP when grown under irrigated conditions, which was consistent with agronomic performance of the corresponding trait values in the high Δ¹³C progeny; that is, significantly greater than that in the low Δ¹³C. Therefore, selection for Δ¹³C was beneficial in increasing grain yield in rain‐fed environments.     

Effects of fertilization on leaf photosynthetic characteristics and grain yield in tartary buckwheat Yunqiao1

Tartary buckwheat (Fagopyrum tataricum Gaertn) has been praised as one of green foods for humans in the 21_st century. Effects of fertilization on leaf photosynthetic characteristics and grain yield of tartary buckwheat has not been yet reported in detail. Our experiment was set as a split-plot factorial. The main plots and subplots were designed by fertilizer ratio and rate as: NPK 1:1:1 (A1), NPK 1:4:2 (A2), NPK 1:2:3 (A3), and 300 (B1), 450 (B2), and 600 (B3) kg (NPK) ha^–1. Our results showed that the grain yield was significantly and positively correlated with the net photosynthetic rate (P _N), stomatal conductance (g _s), transpiration rate (E), PAR, stomatal limitation value (L_s), chlorophyll content (SPAD value), and leaf area index (LAI), while significantly and negatively correlated with intercellular CO₂ concentration (C _i) and water-use efficiency (WUE). The grain yield, P _N, g _s, E, PAR, L_s, SPAD, and LAI increased and then decreased with enhanced fertilization, and their maximum values appeared in the A2B2 treatment. The C _i and WUE decreased and then increased with enhanced fertilization, and their minimum values appeared in the A2B2 treatment. Our results suggested that fertilization had significant effects on the leaf photosynthetic capacity and grain yield of tartary buckwheat Yunqiao 1, and the best fertilization strategy was 450 kg ha^–1 with NPK 1:4:2.     

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