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- Materials and Methods
- Results and Discussion
Stable hydrogen isotope analysis is recognized as a powerful tool in global climate research. Variations in the δ2H and δ18O values of water in ice cores are commonly used to reconstruct past climatic temperature fluctuations, and detailed paleoclimate records were published as early as the 1960s and 1970s (Dansgaard et al., 1969; Epstein et al., 1970)
Furthermore, δ2H values of wood in the annual growth rings of trees can provide the information necessary to reconstruct past climates and to assist in ecophysiological research (Schiegl & Vogel, 1970; Schiegl, 1974; Epstein et al., 1976; Switsur et al., 1996; McCarroll & Loader, 2004; Filot et al., 2006). Site- and compound-specific δ2H values of biomarkers accumulated in sediments are increasingly employed as paleoclimatic and paleohydrological proxies (Sternberg, 1988; Andersen et al., 2001; Sauer et al., 2001; Huang et al., 2002; Sachse et al., 2004).
The general principles governing hydrogen isotopic fractionation are now well delineated. The primary hydrogen source of all organic compounds in the biosphere is water and, in the case of plant biomass, plant leaf water. Hydrogen incorporated into photosynthetic products during primary reduction steps is highly depleted in 2H. However, a significant proportion of these hydrogen atoms are exchanged with hydrogen atoms of water during subsequent metabolism, leading to a secondary enrichment. Hayes (2001) has reviewed fractionation of carbon and, to a lesser extent, hydrogen isotopes in organic compounds produced by a single organism, principally in relation to their enrichment or depletion relative to the total biomass of carbon or hydrogen. This work did not attempt to consider in any depth the isotopic pattern within individual compounds. However, recently an extensive overview and discussion of both intermolecular and intramolecular nonrandom 2H distributions in natural compounds was provided by Schmidt et al. (2003), who also considered their importance in the elucidation of biosynthetic pathways and their potential to assist in assigning an origin to organic compounds in plants.
Natural archives such as ice cores, peatlands and sediments are already widely utilized in climate research, but trees appear to offer an additional very promising method for reconstructing precisely detailed annual climatic histories, not only from living but also from subfossil trees (Schiegl, 1974; Mayr et al., 2003). Indeed, by careful sampling of wood within annual growth rings it may be possible to extract climatic information at a much higher temporal resolution (Barbour et al., 2002; Loader et al., 1995). Early studies on tree rings analysed whole wood but, when Epstein et al. (1976) and Wilson & Grinsted (1977) demonstrated that the wood components lignin, cellulose and hemicellulose differed significantly in isotopic composition, investigations focused on cellulose. One of the main advantages of using cellulose is that it is measured as cellulose nitrate and entirely reflects nonexchangeable hydrogen in this plant component (Epstein et al., 1976). From that time forward, many researchers settled on nonexchangeable hydrogen in cellulose as the best proxy for source water. However, a general problem associated with the determination of the δ2H values of marker compounds for the study of climate and environmental conditions, as well as for investigation of food authenticity investigations, is the isolation of the pure compound for analysis by isotope ratio mass spectrometry (IRMS). Exploitation of components of wood as markers, in particular, has been restricted by the very labour-intensive and time-consuming preparation of samples (e.g. cellulose nitrate). Any improvements to the efficiency of sample preparation would be of immense value as these would not only allow an increase in the number of sampling points within an individual series but also permit replication of time series. Ideally, for accurate determination of the hydrogen isotope signature, the following criteria should apply to the compound or the chemical moiety within a compound:
hydrogen atoms that are nonexchangeable throughout the sample history and during sample preparation and analysis, so that the isotope signature measured reflects the pristine isotopic fractionation of the compound;
high natural abundance in samples;
simple extraction method;
rapid and straightforward sample preparation;
rapid and reliable analysis of the compound;
no isotopic fractionation during any stage of sample processing or analysis.
On the basis of these criteria, we suggest that for 2H analysis of wood the methoxyl groups of lignin offer great potential as target chemical moieties. Lignin, a major component of wood (up to 31%), is produced by secondary metabolic processes and laid down in cellulose cell walls, imparting strength and rigidity to the structure. It can be considered a polymer of three different precursors, termed monolignols, that differ in the degree of methylation of the aromatic ring. The monolignols are ρ-coumaryl alcohol, which has no methoxyl groups, coniferyl alcohol, which has a methoxyl group attached to C-3 of the aromatic ring, and synapyl alcohol, which has methoxyl groups attached to both C-3 and C-5 of the aromatic ring. For more information on lignin structures and biosynthesis, we refer readers to the review by Boerjan et al. (2003). Overall, methoxyl groups can constitute up to 20% of lignin and whole wood can possess up to 6% methoxyl content. Methylation of hydroxyl groups attached to the aromatic ring is catalysed by O-methyltransferases using S-adenosylmethionine (SAM) as the methyl donor. This biochemical origin from SAM has interesting consequences as regards the isotopic composition of the methoxyl groups of lignin. Recent work has shown that the methoxyl groups of lignin and pectin, which together constitute the bulk of the C1 plant pool, have a carbon isotope signature significantly depleted in 13C (Keppler et al., 2004). The depletion between bulk plant biomass and plant methoxyl pools ranges from –11 to –46‰, with the pectin C1 pool generally more depleted than the lignin C1 pool.
Lignin methoxyl groups are considered to be stable; that is, the hydrogen atoms of the methoxyl moiety do not exchange with those of plant water during ongoing metabolic reactions in the plant. Thus, the initial δ2H value of the methoxyl groups of lignin in woody tissue at formation is retained throughout the lifetime of the tree and in preserved tissue. The methoxyl content of lignin in wood is usually determined by the Zeisel method (Zeisel, 1885) using the reaction between methyl ethers and hydroiodic acid (HI) to form methyl iodide (CH3I). Exploiting this reaction (Fig. 1) for the measurement of δ2H values of lignin methoxyl groups ensures that the isotope signal is preserved throughout the analytical procedure, as no isotopic exchange occurs between the methyl groups and HI, and no isotopic fractionation in the course of CH3I formation is observed. In this paper, we report measurements of δ2H values of both whole wood and lignin methoxyl groups from wood samples sourced from various geographical locations which establish a relationship between their isotopic signatures and that of the local precipitation. We demonstrate that the methoxyl groups of lignin meet all the criteria listed above for ideal target chemical moieties for stable hydrogen isotope measurements in wood.