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- Supporting Information
Photosynthetic CO2 fixation discriminates against 13C, so that organic matter formed by plants is naturally 13C-depleted compared with atmospheric CO2 (δ13C = −8‰) (Troughton et al., 1974; O’Leary et al., 1992). However, the isotope fractionation associated with net CO2 assimilation differs between CO2 assimilation pathways: C3 plants have typical δ13C values between −22‰ and −30‰, C4 plants between −9‰ and −13‰, and Crassulacean acid metabolism (CAM) plants between −10‰ and −20‰ (for a review, see Farquhar et al., 1989). The photosynthetic 13C/12C isotope fractionation in plants is mainly caused by diffusional and enzymatic effects, and models based on photosynthetic CO2 fixation have been developed to explain the isotopic composition in primary plant organic matter (Brugnoli & Farquhar, 2000). However, further fractionations may occur after carbon fixation (so-called post-photosynthetic fractionations), thereby changing the isotopic signal of net fixed carbon and causing differences in the isotope composition between plant organs and/or plant organic compounds (Cernusak et al., 2009).
For instance, leaves of C3 plants are systematically 13C-depleted compared with sink organs such as roots or stems (Badeck et al., 2005). This difference between organs is not yet clearly understood, although several reasons have been discussed recently (Badeck et al., 2005; Cernusak et al., 2009). Furthermore, within a single plant organ, quite large differences in δ13C values occur between metabolites. Typically, lipids and lignin are 13C-depleted by up to 7‰ compared with carbohydrates (sucrose, starch) (Park & Epstein, 1961; Winkler et al., 1978). Such differences in δ13C are caused by enzymatic isotope effects (for a review, see Schmidt & Gleixner, 1998) in which the following are well recognized: the oxidation of pyruvate to acetyl-CoA by pyruvate dehydrogenase fractionates against 13C, leading to 13C-depleted acetyl-CoA, which is, in turn, consumed to synthesize fatty acids (Melzer & Schmidt, 1987); the condensation of triose phosphates to fructose-1,6-bisphosphate favours 13C (Gleixner & Schmidt, 1997), thereby explaining the 13C enrichment in the C-3 and C-4 positions of glucose (Rossmann et al., 1991; Gilbert et al., 2009). This isotope effect pervades metabolism, and probably explains the 13C enrichment in leaf-evolved CO2 and in transitory starch (Tcherkez et al., 2004). Positional intramolecular isotope effects of enzymes are thus critical for the 13C distribution within plants and the isotopic composition of CO2 evolved by plant respiration. In other words, enzymatic isotope effects and the intramolecular δ13C values in metabolites will dictate the isotopic CO2 exchange fluxes between plants and the atmosphere (Yakir & Wang, 1996; Barbour & Hanson, 2009).
It has long been recognized that, in order to understand isotope fractionation in plant metabolism, an analysis of isotope redistributions at the intramolecular level is required. This has, however, proved challenging, and relatively few compound-specific δ13C values in metabolites (Collister et al., 1994; Schmidt et al., 2004) and enzymatic isotope effects (Tcherkez & Farquhar, 2005; Mauve et al., 2009) have been investigated. Mostly, only data for whole-molecule δ13C values (δ13Cg, see Table 1 for definitions) have been obtained by isotope ratio mass spectrometry (IRMS), a technique that does not give direct access to the positional δ13C values (δ13Ci). These can only be accessed by IRMS via complex and tedious chemical degradations. The most complex example of this approach is that of Rossmann et al. (1991), who used IRMS following a series of chemical and biochemical degradations to measure the intramolecular δ13C values in glucose of C3 (obtained by the hydrolysis of sucrose in beet syrup) and C4 (from starch of maize flour) origins. Although this work clearly showed the nonuniform 13C distribution in natural glucose with a good precision, the use of this technique is impractical for large sample sets.
Table 1. Symbols used in this article
|δ13C||Carbon isotope composition: carbon isotopic ratio of the molecule relative to the international standard (Vienna Pee Dee Belemnite, V-PDB)|
|δ13Cg||13C mean isotopic composition of a whole molecule measured by isotope ratio mass spectrometry (IRMS)|
|δ13Ci||13C isotopic composition of the carbon position i measured by 13C NMR|
|fi||Molar fraction for a carbon site i measured by 13C NMR = area of the peak corresponding to the carbon position i divided by the sum of all the carbon sites of the molecule ()|
|Fi||Statistical molar fraction for a carbon site i: molar fraction for the carbon site i in the case of a homogeneous 13C distribution within the molecule (Fi = 1/6 for glucose and fructose derivatives)|
|Ai||Isotopic abundance for a carbon site i|
|Ag||Isotopic abundance of a whole molecule|
Quantitative NMR, however, offers the possibility to determine intramolecular δ13Ci values at natural abundance. It analyses directly the target molecule without the need for prior chemical degradation. Site-specific natural isotopic fractionation studied by NMR (SNIF-NMR) was developed for δ2H determinations in the 1980s and is now routinely used for metabolic and climatic analyses and as a tool in authentication (Martin et al., 1986; Remaud et al., 1997; Augusti et al., 2008). However, the use of NMR for the study of intramolecular 13C distributions presents more of a challenge because the range of isotopic variation in natural compounds is c. 10-fold less for 13C than for 2H (c. 50‰ and 500‰, respectively, on the δ scale). Hence, isotopic 13C NMR requires 10 times higher precision. Furthermore, for the effective use of quantitative 13C NMR at natural abundance, a protocol for efficient proton decoupling of 13C–1H interactions is required, as was a means to reduce the extended analytical duration. These obstacles have been overcome by the use of adiabatic decoupling sequences (Tenailleau & Akoka, 2007) and relaxation agents (Caytan et al., 2007b), respectively. Using these protocols, the intramolecular δ13C distributions in vanillin (Caytan et al., 2007a), paracetamol (Silvestre et al., 2009) and natural glucose (Gilbert et al., 2009) have been established recently.
However, the analysis of carbohydrates by 13C NMR is still impeded by isomerization equilibria and inadequate separation of the isomers. In both glucose and fructose, two configurational changes may occur in solution (Fig. 1): mutarotation (α/β forms) and C6 vs C5 cyclization (pyranose/furanose forms) (Flood et al., 1996; Yamabe & Ishikawa, 1999). The isomers have different chemical shifts that generate complex spectra, such that quantitative 13C NMR cannot be carried out reliably with isomeric mixtures. We have previously developed a methodology based on the use of acetyl-isopropylidene derivatives to prevent isomeric interconversions, and this has been applied to glucose analysis (Gilbert et al., 2009). However, the same methodology is insufficient to determine the intramolecular δ13C in fructose. First, fructose forms two products when reacting with iodine in acetone (first step of the derivatization): DAFP (2,3;4,5-di-O-isopropylidene-β-d-fructopyranose) and DAFP-1,2 (1,2;4,5-di-O-isopropylidene-β-d-fructopyranose) (Fig. 2) (Verhart et al., 1992; Kang et al., 1995). The former, the major product, is that desired for 13C NMR analyses, but the formation of the byproduct (DAFP-1,2) may be associated with isotope fractionation and so potentially might distort the δ13C value of DAFP. Second, the product of the second step of the derivatization (1-O-acetyl-2,3;4,5-di-O-isopropylidene-β-d-fructopyranose, MADAFP) has several carbon atoms with similar chemical groups (C-3, C-4 and C-5), giving inadequate resolution in the 13C NMR spectra.
Figure 1. Mutarotational equilibrium of d-fructose involving the linear form, α/β and pyranose/furanose configurations. Numbers in parentheses represent the proportion of each form for d-fructose in aqueous solution at room temperature.
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Figure 2. Procedure for the derivatization of fructose and glucose into 1-O-acetyl-2,3;4,5-di-O-isopropylidene-β-d-fructopyranose (MADAFP) and 3,5,6-triacetyl-1,2-Ο-isopropylidene-α-d-glucofuranose (TAMAGF) for analysis by isotopic 13C NMR. In the first step, an isomer of 2,3;4,5-di-O-isopropylidene-β-d-fructopyranose (DAFP) is obtained (1,2;4,5-di-O-isopropylidene-β-d-fructopyranose, DAFP-1,2), which can be separated from the product DAFP by a selective hydrolysis using 80% aqueous acetic acid (step 2). See text for other abbreviations.
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Here, we describe a method for the determination of δ13Ci in fructose and apply it to obtain the intramolecular 13C patterns in both the fructosyl and glucosyl moieties obtained from sucrose.