A Spectrophotometric Assay for the Enzymatic Demethoxylation of Pectins and the Determination of Pectinesterase Activity

Mangos, Thomas J.; Haas, Michael J.

doi:10.1006/abio.1996.9908

Cited by 17 publications

(4 citation statements)

References 9 publications

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“…The amount of methanol produced in transgenic plants may not be too high to affect plants cell environment. Moreover, knocking down of PME expression in transgenic tomato reportedly does not affect fruit yield or vegetative growth [32], suggesting that the level of methanol content may not affect the plant growth and development. Dwarfism has been reported in transgenic tobacco plants expressing PME which has been attributed to decreased expression level of genes involved in cell wall metabolism viz., endo-1,4-ß-glucanases (Cel2, Cel4, Cel5, Cel7, Cel8), Cellulose synthase (celsyn), endo-xyloglucan transferase (Xytr) and Expansin (NtExp1) [27].…”

Section: Discussionmentioning

confidence: 99%

Enhanced Methanol Production in Plants Provides Broad Spectrum Insect Resistance

et al. 2013

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Plants naturally emit methanol as volatile organic compound. Methanol is toxic to insect pests; but the quantity produced by most of the plants is not enough to protect them against invading insect pests. In the present study, we demonstrated that the over-expression of pectin methylesterase, derived from Arabidopsis thaliana and Aspergillus niger, in transgenic tobacco plants enhances methanol production and resistance to polyphagous insect pests. Methanol content in the leaves of transgenic plants was measured using proton nuclear spectroscopy (1H NMR) and spectra showed up to 16 fold higher methanol as compared to control wild type (WT) plants. A maximum of 100 and 85% mortality in chewing insects Helicoverpa armigera and Spodoptera litura larvae was observed, respectively when fed on transgenic plants leaves. The surviving larvae showed less feeding, severe growth retardation and could not develop into pupae. In-planta bioassay on transgenic lines showed up to 99 and 75% reduction in the population multiplication of plant sap sucking pests Myzus persicae (aphid) and Bemisia tabaci (whitefly), respectively. Most of the phenotypic characters of transgenic plants were similar to WT plants. Confocal microscopy showed no deformities in cellular integrity, structure and density of stomata and trichomes of transgenic plants compared to WT. Pollen germination and tube formation was also not affected in transgenic plants. Cell wall enzyme transcript levels were comparable with WT. This study demonstrated for the first time that methanol emission can be utilized for imparting broad range insect resistance in plants.

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Section: Discussionmentioning

confidence: 99%

Enhanced Methanol Production in Plants Provides Broad Spectrum Insect Resistance

et al. 2013

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“…Methanol concentration in the growth medium was measured according to Mangos and Haas [39] with a minimum of 3 replicates. The method used alcohol oxidase (EC 1.1.3.13), peroxidase (EC 1.11.1.7) and 2,2′-azinobis-(3-ethylbenzthiazoline-6- sulfonic acid) (ABTS) (Calbiochem, EMD Biosciences, Gibbstown, NJ) to determine methanol concentrations.…”

Section: Methodsmentioning

confidence: 99%

A Systems Biology Approach Uncovers Cellular Strategies Used by Methylobacterium extorquens AM1 During the Switch from Multi- to Single-Carbon Growth

et al. 2010

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BackgroundWhen organisms experience environmental change, how does their metabolic network reset and adapt to the new condition? Methylobacterium extorquens is a bacterium capable of growth on both multi- and single-carbon compounds. These different modes of growth utilize dramatically different central metabolic pathways with limited pathway overlap.Methodology/Principal FindingsThis study focused on the mechanisms of metabolic adaptation occurring during the transition from succinate growth (predicted to be energy-limited) to methanol growth (predicted to be reducing-power-limited), analyzing changes in carbon flux, gene expression, metabolites and enzymatic activities over time. Initially, cells experienced metabolic imbalance with excretion of metabolites, changes in nucleotide levels and cessation of cell growth. Though assimilatory pathways were induced rapidly, a transient block in carbon flow to biomass synthesis occurred, and enzymatic assays suggested methylene tetrahydrofolate dehydrogenase as one control point. This “downstream priming” mechanism ensures that significant carbon flux through these pathways does not occur until they are fully induced, precluding the buildup of toxic intermediates. Most metabolites that are required for growth on both carbon sources did not change significantly, even though transcripts and enzymatic activities required for their production changed radically, underscoring the concept of metabolic setpoints.Conclusions/SignificanceThis multi-level approach has resulted in new insights into the metabolic strategies carried out to effect this shift between two dramatically different modes of growth and identified a number of potential flux control and regulatory check points as a further step toward understanding metabolic adaptation and the cellular strategies employed to maintain metabolic setpoints.

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“…By including horseradish peroxidase and a suitable chromogenic peroxidase substrate, such as 2,2‘-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), the amount of H 2 O 2 produced by the AO reaction can be quantified. This AO/peroxidase/ABTS system has been used by several workers as a way to quantify methanol ( , ). However, the presence of antioxidants such as ascorbic acid, which are commonly present in plant extracts, will interfere with this assay by competing for the H 2 O 2 .…”

Section: Introductionmentioning

confidence: 99%

Comparison of Three Colorimetric Reagents in the Determination of Methanol with Alcohol Oxidase. Application to the Assay of Pectin Methylesterase

Anthon

Barrett

2004

J. Agric. Food Chem.

150

106

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Three colorimetric reagents for the determination of formaldehyde, the Nash reagent (ammonia plus acetylacetone), Purpald (4-amino-3-hydrazino-5-mercapto-1,2,4-triazole), and N-methylbenzothiazolinone-2-hydrazone (MBTH), were compared for the determination of methanol when used in conjunction with alcohol oxidase. The combination of alcohol oxidase plus the commonly used Nash reagent was specific for methanol versus ethanol, but had the lowest sensitivity of the three reagents tested. Substituting Purpald for the Nash reagent increased the sensitivity 3-fold while still maintaining a high (59-fold) selectivity for methanol versus ethanol. Using MBTH increased the sensitivity still further, but with a loss of the selectivity toward methanol. Since MBTH reacted with aldehydes under neutral conditions, it could be included along with the alcohol oxidase to act as an aldehyde trap. This prevented further oxidation reactions by alcohol oxidase and allowed for extended incubations. A procedure for assaying low levels of pectin methylesterase activity that relies on this trapping ability is described. In addition, alcohol oxidase plus Purpald is shown to be a simple and sensitive way to measure the methanol released from plant material following the thermal activation of endogenous pectin methylesterase.

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A Spectrophotometric Assay for the Enzymatic Demethoxylation of Pectins and the Determination of Pectinesterase Activity

Cited by 17 publications

References 9 publications

Enhanced Methanol Production in Plants Provides Broad Spectrum Insect Resistance

Enhanced Methanol Production in Plants Provides Broad Spectrum Insect Resistance

A Systems Biology Approach Uncovers Cellular Strategies Used by Methylobacterium extorquens AM1 During the Switch from Multi- to Single-Carbon Growth

Comparison of Three Colorimetric Reagents in the Determination of Methanol with Alcohol Oxidase. Application to the Assay of Pectin Methylesterase

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