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Preparation of starch and other carbon fractions from higher plant leaves for stable carbon isotope analysis

Authors

  • Wolfgang Wanek,

    1. Chemical Physiology of Plants, Institute of Ecology and Conservation Biology, University of Vienna, Althanstrasse 14, A-1090 Vienna, Austria
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  • Sonja Heintel,

    1. Chemical Physiology of Plants, Institute of Ecology and Conservation Biology, University of Vienna, Althanstrasse 14, A-1090 Vienna, Austria
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  • Andreas Richter

    Corresponding author
    1. Chemical Physiology of Plants, Institute of Ecology and Conservation Biology, University of Vienna, Althanstrasse 14, A-1090 Vienna, Austria
    • Institute of Ecology and Conservation Biology, University of Vienna, Althanstrasse 14, A-1090 Vienna, Austria.
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Abstract

The measurement of the carbon isotope composition of starch and cellulose still relies on chemical isolation of these water-insoluble plant constituents and subsequent elemental analysis by isotope ratio mass spectrometry (EA/IRMS) of the purified fractions, while δ13C values of low-molecular-weight organic compounds are now routinely measured by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). Here we report a simple and reliable method for processing milligram quantities of dried plant material for the analysis of the carbon isotope composition of lipids, soluble sugars, starch and cellulose from the same sample. We evaluated three different starch preparation methods, namely (1) enzymatic hydrolysis by α-amylase, (2) solubilization by dimethyl sulfoxide (DMSO) followed by precipitation with ethanol, and (3) partial hydrolysis by HCl followed by precipitation of the resulting dextrins by ethanol. Starch recovery for three commercially available native starches (from potato, rice and wheat) varied from 48 to 81% for the techniques based on precipitation, whereas the enzymatic technique exhibited yields between 99 and 105%. In addition, the DMSO and HCl techniques introduced a significant 13C fractionation of up to 1.9‰, while the carbon isotope composition of native starches analyzed after enzymatic digestion did not show any significant difference from that of untreated samples. The enzymatic starch preparation method was then incorporated into a protocol for determinination of δ13C signatures of lipids, soluble carbohydrates, starch and crude cellulose. The procedure is based on methanol/chloroform/water extraction of dried and ground leaf material. After recovery of the chloroform phase (lipid fraction), the methanol/water phase was deionized by ion exchange (soluble carbohydrate fraction) and the pellet treated with heat-stable α-amylase (starch fraction). The remaining insoluble material was subjected to solvolysis by diglyme (cellulose fraction). The method was shown to be applicable to foliar tissues of a variety of different plant species (spruce, erect brome, maize and soybean). Copyright © 2001 John Wiley & Sons, Ltd.

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