Biosynthetic and environmental effects on the stable carbon isotopic compositions of anteiso- (3-methyl) and iso- (2-methyl) alkanes in tobacco leaves

Abstract

Nicotiana tabacum is the only plant known to synthesise large quantities of anteiso- (3-methyl) alkanes and iso- (2-methyl) alkanes. We investigated the carbon isotope ratios of individual long-chain n-alkanes, anteiso- and iso-alkanes (in the C29-C33 carbon number range) extracted from tobacco grown in chambers under controlled conditions to confirm the pathway used by the tobacco plant to synthesise these particular lipids and to examine whether environmental data are recorded in these compounds. Tobacco was grown under differing temperatures, water availabilities and light intensities in order to control its stable carbon isotope ratios and evaluate isotopic fractionations associated with the synthesis of these particular lipids. The anteiso-alkanes were found to have a predominant even-carbon number distribution (maximising at C32), whereas the iso-alkanes exhibit an odd-carbon number distribution (maximising at C31). Iso-alkanes were relatively more abundant than the anteiso-alkanes and only two anteiso-alkanes (C30 and C32) were observed. The anteiso-alkanes and iso-alkanes were found to be enriched in 13C by 2.8-4.3‰ and 0-1.8‰ compared to the n-alkanes, respectively, consistent with different biosynthetic precursors. The assumed precursor for the odd-carbon-numbered iso-alkanes is iso-butyryl-CoA (a C4 unit derived from valine) followed by subsequent elongation of C2 units and then decarboxylation. The assumed precursor for even-carbon-numbered anteiso-alkanes is α-methylbutyryl-CoA (a C5 unit derived from isoleucine) and subsequent elongation by C2 units followed by decarboxylation. The ratio of carbon atoms derived from α-methylbutyryl-CoA and subsequent C2 units (from malonyl-CoA) is 1:5 for the biosynthesis of a C30 anteiso-alkane. The ratio of carbon atoms derived from iso-butyryl-CoA and subsequent C2 units (from malonyl-CoA) is 4:25 for the synthesis of a C29 iso-alkane. An order of 13C depletion n-alkanes > iso-alkanes > anteiso-alkanes is evident from compound specific isotope data. This trend can probably be attributed to the ratio of the two different sources of carbon atoms in the final wax components. Higher water availability generally results in more depleted stable carbon isotope ratios due to maximised discrimination during carboxylation, associated with less diffusional limitation. This was confirmed in the present study by compound specific isotope analyses of iso-alkanes, anteiso-alkanes and n-alkane lipids extracted from the tobacco leaves. Likewise, light intensity has been shown to influence plant bulk δ13C in previous studies. The carbon isotope ratios of n-alkanes in tobacco grown under low-light conditions were about 2‰ more depleted in 13C than those of lipids extracted from tobacco grown under elevated light conditions. A similar order of difference is observed for the iso-alkanes and anteiso-alkanes (1.8‰ and 1.9‰, respectively). A negligible depletion in carbon isotope ratios was observed for the iso-alkanes and anteiso-alkanes extracted from tobacco grown under elevated temperatures. These results are consistent with the work of Farquhar [Farquhar, G.D., 1980. Carbon isotope discrimination by plants: effects of carbon dioxide concentration and temperature via the ratio of intercellular and atmospheric CO2 concentrations. In: Pearman, G.I. (Ed.), Carbon Dioxide and Climate: Australian Research. Springer, Berlin, pp. 105-110] where temperature appears to have only a minor effect on plant bulk δ13C. Show more