Browsing by Subject "GC-MS"

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    Analysis of ergosterol in single kernel and ground grain by gas chromatography-mass spectrometry
    (Journal of Agricultural and Food Chemistry, 2006-05-11) Steffenson, Brian; Dong, Yanhong; Mirocha, Chester J
    A method for analyzing ergosterol in a single kernel and ground barley and wheat was developed using gas chromatography−mass spectrometry (GC-MS). Samples were saponified in methanolic KOH. Ergosterol was extracted by “one step” hexane extraction and subsequently silylated by N-trimethylsilylimidazole/trimethylchlorosilane (TMSI/TMCS) reagent at room temperature. The recoveries of ergosterol from ground barley were 96.6, 97.1, 97.1, 88.5, and 90.3% at the levels of 0.2, 1, 5, 10, and 20 μg/g (ppm), respectively. The recoveries from a single kernel were between 93.0 and 95.9%. The precision (coefficient of variance) of the method was in the range 0.8−12.3%. The method detection limit (MDL) and the method quantification limit (MQL) were 18.5 and 55.6 ng/g (ppb), respectively. The ergosterol analysis method developed can be used to handle 80 samples daily by one person, making it suitable for screening cereal cultivars for resistance to fungal infection. The ability for detecting low levels of ergosterol in a single kernel provides a tool to investigate early fungal invasion and to study mechanisms of resistance to fungal diseases.
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    Identification of pyruvate decarboxylase/indole pyruvate decarboxylase gene family members from Arabidopsis thaliana.
    (2009-09) Ye, Songqing
    Several biologically important and diverse reactions are regulated by thiamine pyrophosphate (TPP) cofactor-dependent metabolic enzymes, including pyruvate decarboxylase (PDC), indole pyruvate decarboxylase (IPDC), and acetohydroxy acid synthase. PDC is a critical enzyme in plant metabolism that regulates energy production especially during periods of anaerobic stress. IPDC has long been proposed as a key enzyme in the biosynthesis of the plant hormone indole-3-acetic acid (IAA) from tryptophan. Six putative Arabidopsis thaliana PDC gene family members have been individually cloned and expressed in E. coli, and recombinant PDC proteins were purified and biochemically characterized. AtPDC2 was identified as a unique functional PDC based on its measured biochemical activity. The pH and temperature optima for the recombinant protein were 6.2 and 55°C, respectively, and the Km was 3.5 mM. Also, addition of 0.5 mM TPP and 5 mM Mg2+ resulted in the highest activity. However, AtPDC2 lacked any measurable IPDC activity as determined by gas chromatography-mass spectrometry (GC-MS)-based methods. Thus, this mono-functional PDC was different from the more thoroughly studied microbial PDCs, which all have bi-functional activity toward both indole-3-pyruvate and pyruvate substrates. These findings suggest a potential regulatory role for the catalytically inactive PDC proteins in modulation of PDC activity, similar to a mechanism proposed for yeast. None of the pdc mutants showed a change in resistance to chlorsulfuron or imazamox herbicides, and this result was also consistent with the hypothesis that the inactive AtPDC genes may play a role in PDC activity regulation in Arabidopsis. Studies presented here show that the genes most likely to encode proteins with PDC activity or IPDC activity, the PDC gene family, all lack IPDC activity and all except one lack PDC activity. Furthermore, all Arabidopis PDC T-DNA insertion mutants were found to share the same shade avoidance phenotype to as did wild-type plants. These findings bring into question the physiological significance of the IPA pathway for auxin biosynthesis as has been previously proposed. Very low levels of IPDC activity are difficult to measure using procedures developed for the enzyme activity of proteins from bacteria, which produce substantial levels of indole acetaldehyde (IAAld) from indole-3-pyruvate (IPA). To determine the potential activity of plant enzymes, either expressed in E. coli or extracted from Arabidopsis plants, GC-MS assay methods were developed with high sensitivity and specificity. For expressed proteins, IAAld produced from IPA was measured directly using indole carboxaldehyde as an internal standard. This procedure failed, however, to detect IPA in the presence of plant protein extracts; thus, a coupled in vitro reaction with aldehyde dehydrogenase that produced IAA from IPA was developed, and the IAA was quantified using [13C6]IAA as an internal standard, methylation with diazomethane, and GC-MS detection. Together, these methods provide important sensitive and precise methods for the search for IPDC activity in the plant kingdom.
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    Studies of indole-3-butyric acid to indole-3-acetic acid conversion in hazelnut shoot tissuee
    (2015-01) Kreiser, Molly A.
    Indole-3-butyric acid (IBA) is an endogenous compound that appears to regulate both lateral and adventitious root formation in many plant species and is also the auxin most available commercially for application to promote rooting. IBA is converted to indole-3-acetic acid (IAA) by beta oxidation in the peroxisomes. This process has been observed in a number of plant species and has been shown to be critical for normal root development in response to treatment with IBA. In this thesis, the process was investigated in hybrid hazelnuts (C. americana x C. avellana) and American elm (Ulmus americana), in which adventitious rooting is a major bottleneck for vegetative propagation and the efficacy of IBA treatment is highly variable across different cultivars. Using differentially stable isotope labeled IBA and IAA tracer and internal standard, respectively, and using gas chromatography coupled with selected reaction monitoring mass spectrometry, IBA-derived IAA was measured in shoot tissue treated with stable isotope labeled IBA. Variable levels of IBA-to-IAA conversion were observed across different hybrid hazelnut genotypes, which may partially explain differences in rooting ability. In elm, higher levels of IBA-to-IAA conversion were observed in cultivars which formed adventitious roots most easily in softwood stem cutting trials. High rates of root formation is a key trait for establishment of large-scale production systems. Screening for optimal rates of IBA-to-IAA conversion may facilitate selection against genotypes which respond poorly to exogenous IBA. Such genotypes are difficult to propagate using hormone treatment and thus can be eliminated from further evaluations.