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.

Is there any 12C/13C fractionation during starch remobilisation and sucrose export in potato tubers?

“…Finally, after centrifugation at 14 000 g for 15 min at 58C, the supernatant was removed and precipitated starch was freeze-dried for isotopic analysis. While we recognise that some experiments on either natural samples or a mix of commercial products showed that metabolites other than starch might be hydrolysed using HCl, 23 no isotope fractionation was observed here using the HCl method on pure starch samples (see also Ref. 16).…”

Changes in 13C/12C of oil palm leaves to understand carbon use during their passage from heterotrophy to autotrophy

“…Sucrose could either come from the degradation of starch, located at the top of the trunk, 25 or directly from the trunk apex 3 which showed a high concentration of sucrose compared with glucose and fructose. The higher d 13 C of leaf organic matter (OM) (around À27%, close to the d 13 C value of starch) observed during this stage reinforced the hypothesis that the trunk might supply 13 C-enriched sugars (sucrose and starch are known to be 13 Cenriched compared with OM) 32 to the heterotrophic spears.…”

Carbon allocation and carbon isotope fluxes in the plant-soil-atmosphere continuum: a review

“…The PPP releases 13 C-depleted C-1 atoms of glucose as CO 2 (Dieuaide-Noubhani et al, 1995;Bathellier et al, 2009). Moreover, this decarboxylation reaction fractionates against 13 C by about 9.6 ‰ (kinetic isotope effect; Rendina et al, 1984) or against 12 C by 4 ‰ (equilibrium isotope effect; Rendina et al, 1984).…”

“…This so-called "anaplerotic" supply is assumed to refill the TCA when citrate intermediates of the TCA are used, e.g. for amino acid synthesis (Tcherkez and Hodges, 2008;Bathellier et al, 2009). Net discrimination of PEPc against 12 C of 5.7 ‰ (including the equilibrium hydration of CO 2 ) (Farquhar, 1983), enriches organic matter in 13 C and leaves 13 C-depleted CO 2 molecules behind (Gessler et al, 2009a), as long as malic acid is not immediately decarboxylated again (Cernusak et al, 2009).…”