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

The measurement of the carbon isotope composition of starch and cellulose s till relies on chemical isolation of these water-insoluble plant constituen ts and subsequent elemental analysis by isotope ratio mass spectrometry (EA /IRMS) of the purified fractions, while delta C-13 values of low-molecular- weight organic compounds are now routinely measured by gas chromatography/c ombustion/isotope ratio mass spectrometry (GC/C/IRMS). Here we report a sim ple and reliable method for processing milligram quantities of dried plant material for the analysis of the carbon isotope composition of lipids, solu ble sugars, starch and cellulose from the same sample. We evaluated three d ifferent starch preparation methods, namely (1) enzymatic hydrolysis by ol- amylase, (2) solubilization by dimethyl sulfoxide (DMSO) followed by precip itation with ethanol, and (3) partial hydrolysis by HCl followed by precipi tation of the resulting dextrins by ethanol. Starch recovery for three comm ercially available native starches (from potato, rice and wheat) varied fro m 48 to 81% for the techniques based on precipitation, whereas the enzymati c technique exhibited yields between 99 and 105%. In addition, the DMSO and HCl techniques introduced a significant C-13 fractionation of up to 1.9 pa rts per thousand, while the carbon isotope composition of native starches a nalyzed after enzymatic digestion did not show any significant difference f rom that of untreated samples. The enzymatic starch preparation method was then incorporated into a protocol for determinination of delta C-13 signatu res of lipids, soluble carbohydrates, starch and crude cellulose. The proce dure is based on methanol/chloroform/water extraction of dried and ground l eaf material. After recovery of the chloroform phase (lipid fraction), the methanol/water phase was deionized by ion exchange (soluble carbohydrate fr action) and the pellet treated with heat-stable alpha -amylase (starch frac tion). The remaining insoluble material was subjected to solvolysis by digl yme (cellulose fraction). The method was shown to be applicable to foliar t issues of a variety of different plant species (spruce, erect brome, maize and soybean). Copyright (C) 2001 John Wiley & Sons, Ltd.