59th Annual Report on Research 2014

A valuable set of analytical techniques in organic geochemistry relates to the measurement of the isotopic composition of individual hydrocarbons. Plant waxes are one potential source of hydrocarbons, and are largely comprised of long-chain (>C₂₂) alkanes, fatty acids, and alcohols deriving from a variety of terrestrial sources. During Year 1 (2013-2014), we worked to characterize the underlying biological variability of isotopic compositions of plant wax hydrocarbons from plants grown under a relatively controlled set of conditions. We took three approaches to trying to deconvolve biological sources of variability: 1) examination of the intrinsic biological variability among a group of tropical plants collected at a single location, 2) examination of variability among introgression lines between two closely related species that are adapted to very different hydrological regimes, 3) preliminary work on isolating leaf cutin for comparison of the isotopic composition of cutin monomers to plant waxes.

1) In our first experimental approach, we utilized an opportunity to sample the collections of the National Tropical Botanical Garden (Coconut Grove, Florida). This site contains a wide variety of tropical plants grown in close proximity, and the due to the nature of the sampling location, the taxonomy of each sample was precisely constrained.

We conducted a pilot study sampling thirteen plants from six tropical plant families, and measured the abundance of alkanes ranging in chain length from C₂₅ to C₃₅. Abundances of leaf waxes were normalized to leaf surface area and to dry leaf weight. We also measured the ratios D/H in each of these compounds where abundances were high enough to make a measurement. This analysis revealed that while typically alkanes were depleted in deuterium by about 100 permil relative to standard VMOW, there are stark contrasts in deuterium content among species of plants, and in some cases among chain lengths. We are currently using this data as basis for a mixing model to better understand how alkanes deriving from a mixture of sources can result in variation of isotopic composition of the mixture. In year 2 we are planning a similar study utilizing the tropical plant greenhouse (Climatron) at the Missouri Botanical Garden, which contains 1400 species of tropical species.

2) Upon observing biological variability, we designed a new approach to try to deconvolute underlying sources of biological control on isotopic variability. We examined the isotopic variability among introgression lines of two closely related species: cultivated tomato (Solanum pennellii) and wild tomato (Solanum lycopersicum). These species are ideally suited to the study of wax variability, because they are adapted to extremely different hydrological regimes. S. pennellii is highly adapted to water-replete conditions, having been cultivated by humans under irrigated conditions for several thousand years. S. pennellii is native to the Atacama desert in Peru, and is highly adapted to intense drought. For these reasons, tomato has been a model in the study of the underlying mechanisms of how plants become more drought tolerant. Because leaf waxes are thought to play a role in plant water conservation, these plants make an ideal target for studying biological sources of plant wax structure and isotope variability.

We therefore conducted a greenhouse study in which we grew both S. pennellii and S. lycopersicum under identical water-replete conditions. In addition to these two organisms, we grew 76 introgression lines (ILs) in which sections of the genome of S. lycopersicum have been inserted into S. pennellii. Among the 76 lines, the entire genome of S. lycopersicum is represented in the S. pennellii background. Each IL was grown in quadruplicate, and we continuously monitored temperature, relative humidity, PCO2, and the ¹³C content of CO₂ in the greenhouse. ILs were grown in quadruplicate

To date we have analyzed the ¹³C content of each introgression line. This analysis has shown that there are significant differences among ILs, suggesting that we may be able to identify particular trait loci that correspond with genes conferring physiological effects causing ¹³C differences. The variance among ILs is greater than the difference between the two native strains, suggesting that gene interactions (epistasis) may also play a role in determining ¹³C content. In the coming year, we will analyze the D/H content of the ILs and determine whether there are differences that can be traced to particular genetic loci. We also conducted a field study during the summer of 2013 in which all 76 ILs were planted with a replication of 15. Analysis of material from this planting for ¹³C content and D/H ratios is ongoing and will continue in Year 2 of this project.

3) A set of preliminary analyses was conducted to attempt isolation of cutin from both tropical plant leaves and tomato leaves. Enzymatic methods to isolate cutin were not successful, yielding only minimal quantities of material that upon hydrolysis yielded cutin monomers. We conducted the same method on tomato fruit skins, and analysis by solid state NMR and by GCMS analysis of hydrolysis product confirmed the presence of cutin. This suggested that cutin contents in leaves are minimal in comparison to tomato fruit. In Year 2 we will work to further refine this method, with larger quantities of leaf in the hopes of recovering sufficient cutin for comparison of isotopic composition of leaf cutin monomers to that of waxes.