title: Preparation of starch and soluble sugars of plant material for the analysis of carbon isotope composition: a comparison of methods.
abstract: Starch and soluble sugars are the major photosynthetic products, and their carbon isotope signatures reflect external versus internal limitations of CO(2) fixation. There has been recent renewed interest in the isotope composition of carbohydrates, mainly for use in CO(2) flux partitioning studies at the ecosystem level. The major obstacle to the use of carbohydrates in such studies has been the lack of an acknowledged method to isolate starch and soluble sugars for isotopic measurements. We here report on the comparison and evaluation of existing methods (acid and enzymatic hydrolysis for starch; ion-exchange purification and compound-specific analysis for sugars). The selectivity and reproducibility of the methods were tested using three approaches: (i) an artificial leaf composed of a mixture of isotopically defined compounds, (ii) a C(4) leaf spiked with C(3) starch, and (iii) two natural plant samples (root, leaf). Starch preparation methods based on enzymatic or acid hydrolysis did not yield similar results and exhibited contaminations by non-starch compounds. The specificity of the acidic hydrolysis method was especially low, and we therefore suggest terming these preparations as HCl-hydrolysable carbon, rather than starch. Despite being more specific, enzyme-based methods to isolate starch also need to be further optimized to increase specificity. The analysis of sugars by ion-exchange methods (bulk preparations) was fast but produced more variable isotope compositions than compound-specific methods. Compound-specific approaches did not in all cases correctly reproduce the target values, mainly due to unsatisfactory separation of sugars and background contamination. Our study demonstrates that, despite their wide application, methods for the preparation of starch and soluble sugars for the analysis of carbon isotope composition are not (yet) reliable enough to be routinely applied and further research is urgently needed to resolve the identified problems.
authors: Department of Chemical Ecology and Ecosystem Research, University of Vienna, Althanstrasse 14, A-1090 Wien, Austria. andreas.richter@univie.ac.at
authors: Andreas
authors: A
authors: Richter
authors: Wolfgang
authors: W
authors: Wanek
authors: Roland A
authors: RA
authors: Werner
authors: Jaleh
authors: J
authors: Ghashghaie
authors: Maya
authors: M
authors: Jäggi
authors: Arthur
authors: A
authors: Gessler
authors: Enrico
authors: E
authors: Brugnoli
authors: Elena
authors: E
authors: Hettmann
authors: Sabine G
authors: SG
authors: Göttlicher
authors: Yann
authors: Y
authors: Salmon
authors: Camille
authors: C
authors: Bathellier
authors: Naomi
authors: N
authors: Kodama
authors: Salvador
authors: S
authors: Nogués
authors: Astrid
authors: A
authors: Søe
authors: Fillip
authors: F
authors: Volders
authors: Karin
authors: K
authors: Sörgel
authors: Andreas
authors: A
authors: Blöchl
authors: Rolf T W
authors: RT
authors: Siegwolf
authors: Nina
authors: N
authors: Buchmann
authors: Gerd
authors: G
authors: Gleixner
mesh_terms: D002241:Carbohydrates / Q000737:chemistry* / Q000302:isolation & purification*; D002247:Carbon Isotopes / Q000032:analysis*; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D018514:Plant Structures / Q000737:chemistry; D010944:Plants / Q000737:chemistry*; D012995:Solubility; D013213:Starch / Q000032:analysis* / Q000302:isolation & purification*
chemical_list: D002241:Carbohydrates; D002247:Carbon Isotopes; D013213:Starch