Biosynthetic sources of hydrogen isotope variability in acetogenic lipids are driven by phylogeny in eudicot plants
Authors: Baan Jochem , Holloway-Phillips Meisha , Nelson Daniel B. , de Vos Jurriaan M. , Kahmen Ansgar 
Published: March 2021

&lt;p&gt;Hydrogen stable isotope analyses of plant derived &lt;em&gt;n&lt;/em&gt;&amp;#8209;alkanes have been developed as potential tools for ecological, environmental and palaeoclimatological studies. The hydrogen stable isotope composition (&amp;#948;&lt;sup&gt;2&lt;/sup&gt;H) of source and leaf water influence &lt;em&gt;n&lt;/em&gt;&amp;#8209;alkane &amp;#948;&lt;sup&gt;2&lt;/sup&gt;H values (&amp;#948;&lt;sup&gt;2&lt;/sup&gt;H&lt;sub&gt;alkane&lt;/sub&gt;), but hydrogen isotope fractionation occurs during plant carbon metabolism. Large variation in &amp;#948;&lt;sup&gt;2&lt;/sup&gt;H&lt;sub&gt;alkane&lt;/sub&gt; values among species at a single geographic location have been observed, suggesting that metabolic isotope effects are large, but the origin of this biochemical variation remains poorly understood. To explore the variation in &amp;#948;&lt;sup&gt;2&lt;/sup&gt;H&lt;sub&gt;alkane&lt;/sub&gt;, and test if phylogenetic relatedness structures variation in &amp;#948;&lt;sup&gt;2&lt;/sup&gt;H&lt;sub&gt;alkane&lt;/sub&gt; values across species, we measured &amp;#948;&lt;sup&gt;2&lt;/sup&gt;H values of &lt;em&gt;n&lt;/em&gt;&amp;#8209;alkanes in a total of 218 samples and 183 eudicot plant species grown in a single common garden during the 2019 growing season. Our results show that most variation in &amp;#948;&lt;sup&gt;2&lt;/sup&gt;H&lt;sub&gt;alkane&lt;/sub&gt; values could be explained by family and genus identity, and &amp;#948;&lt;sup&gt;2&lt;/sup&gt;H&lt;sub&gt;alkane&lt;/sub&gt; values significantly evolved along the phylogeny. To identify the intracellular location where the variation in &amp;#948;&lt;sup&gt;2&lt;/sup&gt;H&lt;sub&gt;alkane&lt;/sub&gt; is induced, we measured &amp;#948;&lt;sup&gt;2&lt;/sup&gt;H&lt;sub&gt;alkane&lt;/sub&gt; values as well as &amp;#948;&lt;sup&gt;2&lt;/sup&gt;H values of plastid&amp;#8209;produced precursors, as the acetogenic pathway in higher plants is spatially separated within cells, where fatty acids up to 18 carbon units are produced in the plastid, and additional chain elongation (e.g., for &lt;em&gt;n&lt;/em&gt;&amp;#8209;alkane synthesis) occurs in the endoplasmic reticulum. Specifically, during the 2020 growing season, we measured &amp;#948;&lt;sup&gt;2&lt;/sup&gt;H values of &lt;em&gt;n&lt;/em&gt;-alkanes and &lt;em&gt;n&lt;/em&gt;&amp;#8209;C&lt;sub&gt;16:0&lt;/sub&gt; fatty acids (palmitic acid; &amp;#948;&lt;sup&gt;2&lt;/sup&gt;H&lt;sub&gt;n&amp;#8209;C16:0&lt;/sub&gt;) in a total of 61 samples and 58 eudicot plant species grown in a single common garden and determined how the two spatially separated parts of the acetogenic pathway influenced &amp;#948;&lt;sup&gt;2&lt;/sup&gt;H&lt;sub&gt;alkane&lt;/sub&gt; values by comparing to &amp;#948;&lt;sup&gt;2&lt;/sup&gt;H&lt;sub&gt;n&amp;#8209;C16:0&lt;/sub&gt; values. &amp;#948;&lt;sup&gt;2&lt;/sup&gt;H&lt;sub&gt;n&amp;#8209;C16:0&lt;/sub&gt; and &amp;#948;&lt;sup&gt;2&lt;/sup&gt;H&lt;sub&gt;alkane&lt;/sub&gt; values showed a significant positive correlation, suggesting that &amp;#948;&lt;sup&gt;2&lt;/sup&gt;H&lt;sub&gt;alkane&lt;/sub&gt; biosynthetic isotopic variability is largely shaped by processes in the plastid leading up to the synthesis of palmitic acid. Strikingly, &amp;#948;&lt;sup&gt;2&lt;/sup&gt;H&lt;sub&gt;n&amp;#8209;C16:0&lt;/sub&gt; values were almost always higher than &amp;#948;&lt;sup&gt;2&lt;/sup&gt;H&lt;sub&gt;alkane&lt;/sub&gt; values, and the offset between these values (as an enrichment factor; &amp;#400;&lt;sub&gt;C16:0&amp;#8209;alkane&lt;/sub&gt; in &amp;#8240;) was also related to phylogeny. This illustrates that the extent to which the two spatially separated parts of the acetogenic pathway influence &amp;#948;&lt;sup&gt;2&lt;/sup&gt;H values of &lt;em&gt;n&lt;/em&gt;&amp;#8209;alkanes depends on species identity due to phylogenetic effects. Our results demonstrate that variation in &amp;#948;&lt;sup&gt;2&lt;/sup&gt;H&lt;sub&gt;alkane&lt;/sub&gt; values among species is strongly related to phylogenetic effects, and that most of these effects are generated early in lipid biosynthesis. Future studies should address if the observed variability can be explained by metabolic traits.&lt;/p&gt;