Environmental and biosynthetic influences on carbon and hydrogen isotope ratios of leaf wax n-alkanes

Both carbon and hydrogen isotope ratios of leaf-wax n-alkanes are influenced by the availability of water in a plant's growth environment. Carbon isotope ratios of bulk tissues in C3 plants demonstrate a strong inverse relationship with measures of available moisture (e.g. mean annual precipitation and precipitation/evaporation). Similarly, hydrogen isotope ratios of leaf wax n-alkanes (δD_l) can be enriched relative to precipitation (δD_w) by transpiration, which is related to relative humidity and the leaf-to-air vapor pressure deficit. Thus, D-enrichment of leaf-wax n-alkanes relative to precipitation, termed the apparent fractionation (²ɛ_l/w), becomes more positive with increasing aridity. In theory, more positive values of leaf-wax δ¹³C (δ¹³C_l) and ²ɛ_l/w of leaf-wax n-alkanes should both correspond to more arid conditions in C3 plants. Here we review published and unpublished data on over 100 plants to examine this relationship. Contrary to expectations, C3 dicots show no clear relationship between δ¹³C_l and ²ɛ_l/w. This global lack of correlation is surprising given our understanding of aridity related isotopic effects in C3 plants. One possibility is that the implicit assumption of constant fractionation between lipid and bulk tissue is flawed due to the effects of different biosynthetic carriers and reaction pathways. We explore this possibility by examining the offset of leaf-wax carbon isotopes from the bulk leaf tissue (¹³ɛ_l/bulk). Different offsets would indicate additional biosynthetic processes are affecting δ¹³C_l in addition to any direct effects from aridity. We find that ¹³ɛ_l/bulk is highly variable, ranging from -1 to -16‰, which could explain the lack of correlation between δ¹³C_l and ²ɛ_l/w. In addition, ¹³ɛ_l/bulk values for C3 and C4 monocots (averages of -10.6 and -11.4‰ respectively) represent significantly greater offset between leaf wax and bulk tissue than in C3 dicots (average of -4.3‰), which is consistent with previous studies. Notably, ²ɛ_l/w and ¹³ɛ_l/bulk are positively correlated for all C3 plants, as well as for sub-divided C3 plant groups of dicots, monocots and gymnosperms. The positive relationship of ²ɛ_l/w and ¹³ɛ_l/bulk for C3 plants suggests co-varying influences of environmental properties on biosynthetic fractionation, such as root carbon storage use and water isotopes at the time of lipid synthesis. In contrast, C4 plants showed no correlation between ²ɛ_l/w and ¹³ɛ_l/bulk, which could signify differences in biosynthetic pathways or resource strategies between C3 and C4 plants. Paired leaf-wax carbon and hydrogen isotopic measurements have potential to elucidate isotope effects associated with ecohydrology and biosynthesis, which can strengthen paleoenvironmental interpretations of molecular isotopic data. Our analysis suggests both effects may be important determinants of plant-lipid isotope values.