Preparation of starch and soluble sugars of plant material for analysis of carbon isotope composition: a comparison of methods
Academic Article
Publication Date:
2009
abstract:
Starch and soluble sugars are the major photosynthetic products, and their carbon isotope signatures
reflect external versus internal limitations of CO2 fixation. There has been recent renewed interest in
the isotope composition of carbohydrates, mainly for use in CO2 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; ionexchange
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 C4 leaf spiked with C3 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 HClhydrolysable
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 ionexchange
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.
reflect external versus internal limitations of CO2 fixation. There has been recent renewed interest in
the isotope composition of carbohydrates, mainly for use in CO2 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; ionexchange
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 C4 leaf spiked with C3 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 HClhydrolysable
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 ionexchange
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.
Iris type:
01.01 Articolo in rivista
List of contributors:
Brugnoli, Enrico
Published in: