Syntheses of C-14 labeled methyl malvalates

Malvalic acid, the principle cyclopropene acid in cottonseed oil, has been synthesized with carbon-14 at the 1-position, at the ring methano-position, and at the 10-position. A method for reaching malvalic-9-¹⁴C acid has also been worked out. These labeled compounds should prove useful in studies on the biochemical reactions and degradation of malvalic acid.     

Reduction of cyclopropene by NifV- and wild-type nitrogenases from Klebsiella pneumoniae.

The nitrogenase from wild-type Klebsiella pneumoniae reduces cyclopropene to cyclopropane and propene in the ratio 1:2 at pH 7.5. We show in this paper that the nitrogenase from a nifV mutant of K. pneumoniae also reduces cyclopropene to cyclopropane and propene, but the ratio of products is now 1:1.4. However, both nitrogenases exhibit the same Km for cyclopropene (2.1 x 10(4) +/- 0.2 x 10(4) Pa), considerably more than the Km for the analogous reaction with Azotobacter vinelandii nitrogenase under the same conditions (5.1 x 10(3) Pa). Analysis of the data shows that the different product ratio arises from the slower production of propene compared with cyclopropane by the mutant nitrogenase. During turnover, both nitrogenases use a large proportion of the electron flux for H2 production. CO inhibits the reduction of cyclopropene by both K. pneumoniae proteins, but the mutant nitrogenase exhibits 50% inhibition at approx. 10 Pa, whereas the corresponding value for the wild-type nitrogenase is approx. 110 Pa. However, H2 evolution by the mutant enzyme is much less affected than is cyclopropene reduction. CO inhibition of cyclopropene reduction by the nitrogenases coincides with a relative increase in H2 evolution, so that in the wild-type (but not the mutant) the electron flux is approximately maintained. The cyclopropane/propene production ratios are little affected by the presence of CO within the pressure ranges studied at least up to 50% inhibition.     

Mass spectrometry of derivatives of cyclopropene fatty acids

Diketo fatty acids prepared by ozonization of cyclopropene fatty acids have been separated and purified by chromatographic techniques. Mass spectra of esters of these compounds and of methanethiol adducts of cyclopropene acid esters are reported and interpreted. Location of the ring from examination of mass spectra of these derivatives appears to be a straightforward matter.     

Eriolaena hookeriana seed oil: A rich source of malvalic acid

The seed oil of Eriolaena hookeriana (Sterculiaceae) contains malvalic (25.8%) and sterculic (6.0%) acids in addition to normal fatty acids. Eriolaena hookeriana is the second known species of the Sterculiaceae plant family whose seed oil contains more malvalic acid than sterculic acid. Gas chromatography-mass spectrometry (GS-MS) study of the silver nitrate-methanol treated methyl esters has been carried out for unequivocal characterisation of the individual cyclopropene acids. Genesis of diagnostic fragment ions is discussed.     

Protonation of cyclopropene complexes of platinum(0) and the reduction of cyclopropene by nitrogenases

The reactions of a series of platinum(0) cyclopropene complexes with HCl have been studied, and it is shown that the products include both ring-intact (cyclopropane-type) and ring-opened (propene-type) materials. All products are believed to be formed by hydrocarbon insertions into transient platinum-hydrogen bonds formed by addition of HCl to platinum. Mechanistic interpretation is complicated by group migrations, and double-bond shifts. Oxidative addition of a σ-cyclopropys to platinum plays a part in ring-opening reactions, whereas the cyclopropanesare believed to be produced by further hydrocarbon insertions into transient platinum-hydrogen bonds. The relevance of these models to the mechanism of reduction of cyclopropene by nitrogenases is discussed.     

The effect of chemical structure on the antagonism by cyclopropenes of ethylene responses in banana

The compounds, cyclopropene, 1-methylcyclopropene, 3-methylcyclopropene, 1,3-dimethylcyclopropene, 3,3-dimethylcyclopropene, 1,3,3-trimethylcyclopropene, 3-methyl-3-vinylcyclopropene, and 3-methyl-3-ethynylcyclopropene, and 1,2-dimethylcyclopropene were tested as antagonists to the ethylene receptor in bananas. All of the compounds inactivated the receptor and the bananas did not respond to ethylene even at 1000 nL L^–1. Large differences were found in the concentration required (0.7–20,000 nL L^–1 for 24h) to inactivate the receptor and in the duration of inactivation (3–12 days at 24C depending on the compound). After this time the bananas responded to ethylene and appeared to ripen normally.     

Label-free in situ Imaging of Lignification in Plant Cell Walls

Meeting growing energy demands safely and efficiently is a pressing global challenge. Therefore, research into biofuels production that seeks to find cost-effective and sustainable solutions has become a topical and critical task. Lignocellulosic biomass is poised to become the primary source of biomass for the conversion to liquid biofuels^1-6. However, the recalcitrance of these plant cell wall materials to cost-effective and efficient degradation presents a major impediment for their use in the production of biofuels and chemicals⁴. In particular, lignin, a complex and irregular poly-phenylpropanoid heteropolymer, becomes problematic to the postharvest deconstruction of lignocellulosic biomass. For example in biomass conversion for biofuels, it inhibits saccharification in processes aimed at producing simple sugars for fermentation⁷. The effective use of plant biomass for industrial purposes is in fact largely dependent on the extent to which the plant cell wall is lignified. The removal of lignin is a costly and limiting factor⁸ and lignin has therefore become a key plant breeding and genetic engineering target in order to improve cell wall conversion.Analytical tools that permit the accurate rapid characterization of lignification of plant cell walls become increasingly important for evaluating a large number of breeding populations. Extractive procedures for the isolation of native components such as lignin are inevitably destructive, bringing about significant chemical and structural modifications^9-11. Analytical chemical in situ methods are thus invaluable tools for the compositional and structural characterization of lignocellulosic materials. Raman microscopy is a technique that relies on inelastic or Raman scattering of monochromatic light, like that from a laser, where the shift in energy of the laser photons is related to molecular vibrations and presents an intrinsic label-free molecular "fingerprint" of the sample. Raman microscopy can afford non-destructive and comparatively inexpensive measurements with minimal sample preparation, giving insights into chemical composition and molecular structure in a close to native state. Chemical imaging by confocal Raman microscopy has been previously used for the visualization of the spatial distribution of cellulose and lignin in wood cell walls^12-14. Based on these earlier results, we have recently adopted this method to compare lignification in wild type and lignin-deficient transgenic Populus trichocarpa (black cottonwood) stem wood¹⁵. Analyzing the lignin Raman bands^16,17 in the spectral region between 1,600 and 1,700 cm^-1, lignin signal intensity and localization were mapped in situ. Our approach visualized differences in lignin content, localization, and chemical composition. Most recently, we demonstrated Raman imaging of cell wall polymers in Arabidopsis thaliana with lateral resolution that is sub-μm¹⁸. Here, this method is presented affording visualization of lignin in plant cell walls and comparison of lignification in different tissues, samples or species without staining or labeling of the tissues.Download video file.^{(47M, mov)}     

Wheat bulb fly,Delia coarctata,larval attraction to phenolic components of host‐plant root exudates

Wheat bulb fly (WBF), Delia coarctata Fallén (Diptera: Anthomyiidae), larvae are a subterranean pest of wheat [Triticum aestivum L. (Poaceae)] and other cereals. Larvae locate host plants through chemotaxis and chemokinesis, utilising the primary plant metabolite carbon dioxide as a ‘search trigger’ and Poaceae‐specific secondary plant metabolites exuded from the plant. The aim of this study was to use arena bioassays to identify further compounds involved in the host‐finding process. The larval behavioural response to four concentrations of syringic and vanillic acid, chemical constituents of host‐plant exudates, were tested. Analysis of the final resting position of D. coarctata larvae by the Rayleigh test of uniformity identified attraction to wheat seedling exudates and to both compounds at the lowest concentrations tested, with syringic acid concentrations being most attractive at 0.1 mg l^?1 and vanillic acid being most attractive at 0.001 mg l^?1. These results add more detail to the subterranean chemical ecology of this species, allowing a behavioural sequence for host‐plant orientation by WBF larvae to be proposed.     

Evidence for a nonstatistical carbon isotope distribution in natural glucose

The relative carbon isotope content (δ¹³C value) in each position of glucose from a C₄ plant (maize starch) and a C₃ plant (sugar beet sucrose) has been determined by stepwise chemical and biochemical degradation of the molecule and stable isotope ratio measurement of the fragments. The suitability of the degradation methods has been tested through their chemical yield and isotope balance. The results from both methods agreed perfectly, revealing a defined and reproducible ¹³C distribution in glucose from both origins. Most prominent was a relative ¹³C enrichment by 5 to 6 δ-units in position 4 and a depletion by about 5 δ-units in carbon 6. As possible reasons for these nonstatistical isotope distributions, isotope effects of the aldolase, the triose phosphate isomerase, and the transketolase reactions during carbohydrate biosynthesis are discussed. The practical importance of the results in regard to isotope distributions in secondary plant products as a means for food authenticity control is outlined.     

Ontogenetic and temporal trajectories of chemical defence in a cyanogenic eucalypt

Many studies have shown that similarly aged plants within a species or population can vary markedly in the concentration of defence compounds they deploy to protect themselves from herbivores. Some studies have also shown that the concentration of these compounds can change with development, but no empirical research has mapped such an ontogenetic trajectory in detail. To do this, we grew cyanogenic Eucalyptus yarraensis seedlings from three half-sibling families under constant glasshouse conditions, and followed their foliar cyanogenic glycoside (prunasin) concentration over time for 338 days after sowing (DAS). Plants in all families followed a similar temporal pattern. Plants increased in foliar prunasin concentration from a very low level (10 μg cyanide (CN) equivalents g⁻¹) in their first leaves, to a maximum of, on average, 1.2 mg CN g⁻¹ at about 240 DAS. From 240 to 338 DAS, prunasin concentration gradually decreased to around 0.7 mg CN g⁻¹. Significant differences between families in maximum prunasin concentration were detected, but none were detected in the time at which this maximum occurred. In parallel with these changes in prunasin concentration, we detected an approximately linear increase in leaf mass per unit leaf area (LMA) with time, which reflected a change from juvenile to adult-like leaf anatomy. When ontogenetic trajectories of prunasin against LMA were constructed, we failed to detect a significant difference between families in the LMA at which maximum prunasin concentration occurred. This remarkable similarity in the temporal and ontogenetic trajectories between individuals, even from geographically remote families, is discussed in relation to a theoretical model for ontogenetic changes in plant defence. Our results show that ontogeny can constrain the expression of plant chemical defense and that chemical defense changes in a nonlinear fashion with ontogeny.     

Mass spectrometric analysis of cytokinins in plant tissue VII. Quantification of cytokinin bases by negative ion mass spectrometry

A study of derivatives of N⁶-(isopent-2-enyl)adenine formed by substitution at N-9 indicated that sensitivity of detection by chemical ionization mass spectrometry was maximized by a pentafluorobenzyl substituent and negative ion monitoring. O-t-Butyldimethylsilyl-9-pentafluorobenzyl derivatives of zeatin (Z),cis-zeatin (cis-Z), and dihydrozeatin (DZ) were characterized by mass spectrometry. A procedure was based on these stable derivatives and negative ion chemical ionization mass spectrometry for quantification of zeatin and dihydrozeatin in plant tissue.     

Analytical Procedures for the Sequential Extraction of 14C-Labeled Constituents from Leaves, Bark and Wood of Cottonwood Plants

A procedure for the extraction, separation, and measurement of photosynthetically fixed ¹⁴C in up to 8 chemical fractions (CHCl), amino acids, organic acids, sugars, protein, starch, hemicellulose, and residue) from small samples (1 to 100 mg) of cottonwood (Populus deltoides Bartr.) leaf material is described. The different chemical fractions are extracted in a sequence of chemical, ion exchange, and enzymatic steps. The ¹⁴C-activity in these major fractions is then determined with liquid scintillation spectrometry. These major fractions (e.g., sugars, amino acids, organic acids) can be further separated into their individual chemical components by standard thin-layer or gas- chromatographic methods for quantitative analysis if specific activities are desired. The major advantage of the procedure is that many chemical fractions can be sequentially separated with good reproducibility from a small amount of plant material without transfer of the material from the original homogenizer or centrifuge tube.     

The foliar chemical profile of criollo avocado, Persea americana var. drymifolia (Lauraceae), and its relationship with the incidence of a gall-forming insect, Trioza anceps (Triozidae)

The role of phytochemical diversity in plant defense was studied using the specialist herbivore Trioza anceps Tuthill (avocado gall-forming insect), and the volatile fraction of the foliar chemicals of its host, the creole avocado (Persea americana var. drymifolia (Schect. &; Cham.) Blake). Two hypotheses were tested: a) plant defense is determined by phytochemical diversity, and b) plant defense is determined by single compounds or specific blends of compounds. Simple and multiple regressions and a size and shape analysis (which considers the compounds relative and absolute concentrations within the leaf blend) were used to test these hypotheses. Simple regressions of gall incidence and chemical diversity and the tree origin elevation and latitude were very weak. The linear multiple regression to explain gall incidence with 33 foliar compounds and geographical data produced a model with low predictive power (R² = 0.13). The size and shape analysis showed intraspecific variation in leaf chemical profiles among five tree groups, classified by the number of galls per 10 cm² of leaf. Discriminant analysis separated clearly the tree groups’ chemical profiles through specific compounds. These results suggest that the gall incidence is associated with specific chemical profiles, rather than to high or low foliar phytochemical diversity.     

Mass spectrometric analysis of cytokinins in plant tissue VII. Quantification of cytokinin bases by negative ion mass spectrometry

A study of derivatives of N⁶-(isopent-2-enyl)adenine formed by substitution at N-9 indicated that sensitivity of detection by chemical ionization mass spectrometry was maximized by a pentafluorobenzyl substituent and negative ion monitoring. O-t-Butyldimethylsilyl-9-pentafluorobenzyl derivatives of zeatin (Z),cis-zeatin (cis-Z), and dihydrozeatin (DZ) were characterized by mass spectrometry. A procedure was based on these stable derivatives and negative ion chemical ionization mass spectrometry for quantification of zeatin and dihydrozeatin in plant tissue.For part VI, see Letham and Singh (1989).     

Influence of the Addition of Different Metals to an Activated Sludge System on the Enhanced Biological Phosphorus Removal

Activated sludge systems can be operated to select bacteria which accumulate phosphorus as polyphosphate. By these means, phosphate can be removed without the addition of chemical precipitants. This contribution presents results of experiments with a bench-scale purification plant for domestic sewage. The goal was to find the concentrations of relevant metal cations at which the biological P removal may be affected by competing chemical (precipitation) or physical (adsorption) processes. For this goal, increasing amounts of iron and calcium, respectively, were added into the pilot plant. During the addition of iron, the proportion of chemically (iron-) bound phosphorus increased from 10 to more than 50%. The P-release rate substantially decreased with increasing amount of added iron. An addition of small amounts of iron enhanced the long-term stability of the P elimination as a whole. During the experimental period with addition of calcium, the proportion of Ca-bound phosphorus increased from 1 to 2% to almost 15%. In batch experiments a measurable Ca-phosphate precipitation took place at a pH value of at least 8.0 and a Ca-concentration of at least 100 mg/l. The increase in hardness of the influent waste water didn't produce any positive effect on the stability of the enhanced biological phosphorus removal. The metal ions Ca²⁺, Mg²⁺ and K⁺ serve as counter-ions in the polyhosphate chains. They were identified and quantified by X-ray spectrometry in combination with scanning transmission electron microscopy. A release of Mg²⁺ and K⁺ occured simultaneously with the degration of polyphosphates (PP). The PP bound to Ca was not redox sensitive.     

Influence of cold temperatures on leaf lipids of Hibiscus rosa-sinensis

Triaclyglycerols (TAG) accounted for 15% of the leaf acyl lipids in a Hibiscus rosa-sinensis plant that survived the January 1986 freeze in Florida, U.S.A. This high TAG level suggested that the plant had cold hardened. The TAG concentration in leaves on new shoots from this plant was greater than that in new leaves on new shoots from five plants frozen to the roots during the freeze. However, five months later, the amounts of TAG in the six Hibiscus plants were nearly the same. The minor differences in TAG levels, however, related to the survival rate of these six plants during subsequent freezes. In addition to linoleic acid, two cyclopropene fatty acids, malvalic and sterculic, were major constituents of the leaf TAG. Hibiscus plants placed in environmental chambers under control and cold-hardening regimes optimized for Citrus showed TAG concentrations of 7% in control and 20% in hardened plants. A survey of neutral lipids in Hibiscus and other plants showed that plastoquinone A (B) and α-tocopherol decreased and plastoquinone C increased under cold-hardening conditions. Polyprenols, a major component of Hibiscus leaves under normal conditions, declined greatly under cold-hardening regimes.     

Modification of fatty acid composition of lipid accumulating yeasts with cyclopropene fatty acid desaturase inhibitors

Summary The effect of cyclopropene fatty acids, sterculic and malvalic, on the lipids of yeasts grown under nitrogen limiting, lipid accumulating, conditions was studied. The ratio of stearic to oleic acid showed a dose response effect, with an increase in stearic acid content as the dose of cyclopropene fatty acid increased, and a corresponding reduction in oleic acid. Linoleic and linolenic acids were not affected to the same extent. These effects are shown for the yeasts Candida sp. 107, Trichosporon cutaneum, and Rhodosporidium toruloides.     

Microchemical analysis of laser-microdissected stone cells of Norway spruce by cryogenic nuclear magnetic resonance spectroscopy

Stone cells (sclereids) in Norway spruce (Picea abies) bark have been reported to be highly lignified tissues that are important in physical defence against bark beetle invasion. Microchemical analyses of the low-molecular weight compounds in the stone cells of Norway spruce were carried out using laser microdissection in combination with cryogenic nuclear magnetic resonance and mass spectrometry (LMD/NMR/MS). Two phenolic compounds, the stilbene astringin and the dihydroflavonol dihydroxyquercetin 3′-O-β-d-glucopyranoside, were identified indicating that stone cells are more than just repositories for lignin. Both of these compounds were also found to be present in other phloem tissue at a higher level than in the stone cells based on quantification by cryogenic ¹H NMR. Our results suggest that stone cells may be involved in chemical as well as physical defense against bark beetles and their associated microorganisms. This paper reports on the identification of secondary plant metabolites from a single laser-microdissected population of plant cells offering a sensitive new way to determine the chemical profile of specific plant cell types with a high degree of precision.     

海河流域土壤中磷的库存量与通量研究

本通过对河流域土壤中磷及其形态含量的分析结果,计算出该流域土壤中磷的库存量为６．４２×１０＾１０ｋｇ。并以成土母质,人畜粪,化肥施入量和径流带入量,大气降尘及植物的残留量等因素为对土壤中磷的贡献,同时以径流的带出,大气的飘尘及植物体茎叶等因素为磷的迁出部分,计算出海流域土体中磷的进出量分别为４．２４×１０＾８ｋｇ／ａ和３．６６×１０＾８ｋｇ／ｄ,以此来研究其通量关系。     

Challenging the paradigm of nitrogen cycling: no evidence of in situ resource partitioning by coexisting plant species in grasslands of contrasting fertility

In monoculture, certain plant species are able to preferentially utilize different nitrogen (N) forms, both inorganic and organic, including amino acids and peptides, thus forming fundamental niches based on the chemical form of N. Results from field studies, however, are inconsistent: Some showing that coexisting plant species predominantly utilize inorganic N, while others reveal distinct interspecies preferences for different N forms. As a result, the extent to which hypothetical niches are realized in nature remains unclear. Here, we used in situ stable isotope tracer techniques to test the idea, in temperate grassland, that niche partitioning of N based on chemical form is related to plant productivity and the relative availability of organic and inorganic N. We also tested in situ whether grassland plants vary in their ability to compete for, and utilize peptides, which have recently been shown to act as an N source for plants in strongly N-limited ecosystems. We hypothesized that plants would preferentially use NO₃⁻-N and NH₄⁺-N over dissolved organic N in high-productivity grassland where inorganic N availability is high. On the other hand, in low-productivity grasslands, where the availability of dissolved inorganic N is low, and soil availability of dissolved organic N is greater, we predicted that plants would preferentially use N from amino acids and peptides, prior to microbial mineralization. Turves from two well-characterized grasslands of contrasting productivity and soil N availability were injected, in situ, with mixtures of ¹⁵N-labeled inorganic N (NO₃⁻ and NH₄⁺) and ¹³C¹⁵N labeled amino acid (l-alanine) and peptide (l-tri-alanine). In order to measure rapid assimilation of these N forms by soil microbes and plants, the uptake of these substrates was traced within 2.5 hours into the shoots of the most abundant plant species, as well as roots and the soil microbial biomass. We found that, contrary to our hypothesis, the majority of plant species across both grasslands took up most N in the form of NH₄⁺, suggesting that inorganic N is their predominant N source. However, we did find that organic N was a source of N which could be utilized by plant species at both sites, and in the low-productivity grassland, plants were able to capture some tri-alanine-N directly. Although our findings did not support the hypothesis that differences in the availability of inorganic and organic N facilitate resource partitioning in grassland, they do support the emerging view that peptides represent a significant, but until now neglected, component of the terrestrial N cycle.