Untargeted and Targeted Metabolomics to Understand Plant Growth Regulation and Evolution in Wollemi Pine (wollemia Nobilis)

Wollemi pine (Wollemia nobilis (Jones, Hill, Allen)) is a living fossil, known only through fossil records until its 1994 discovery in Australia. Wollemi Pine is closely related to Norfolk Island pine (Araucaria heterophylla (Salisb.) Franco), making it an interesting system to study metabolic evolution. We employed untargeted liquid chromatography mass spectrometry-based (LC-MS) metabolomics, with chemometrics, pathway analysis, and our novel plant growth regulator (PGR) putative identification tool (HormonomicsDB), to explore the metabolomes of both species. We identified PGR conjugates and found cytokinin, zeatin, and brassinosteroid pathways to be overrepresented in Wollemi pine tissues, suggesting these PGRs play an important role in its survival. Melatonin, a proposed ancient and stress-related PGR, was not identified in untargeted analysis of either species. Based on our untargeted results, we employed targeted LC-MS to quantify brassinolide and confirm the absence or presence of melatonin in Wollemi and Norfolk Island pine. Both species had similar concentrations of brassinolide, while Wollemi pine had significantly higher melatonin levels than Norfolk Island pine. High levels of melatonin and brassinolide in Wollemi pine supports the hypothesis that these are ancient PGRs that conferred an evolutionary advantage in Wollemi, allowing it to persist.