Population genetics of selected Boraginaceae and their transcriptomic changes upon phytohormonal and microbial interaction

The family Boraginaceae includes many medicinal plant species that produce valuable secondary metabolites. The most important secondary metabolites are alkannin, its enantiomer shikonin and their derivatives (A/S), which exclusively accumulate in the root periderm. A/S have been shown to be beneficial for the treatment of humans such as wounds and inflammation, and possess the potential to be used as antibacterial, antiviral and antitumor agents. Like for many other herbal compounds, the main source of A/S are wild plants, which often leads to an overexploitation exerting immense pressure on natural populations. In addition, wild plants suffer from variable yield of A/S, a constraint for subsequent standardized extraction processes aiming at a high yield. In order to aid in conservation programs but also to predict variation in A/S contents, an understanding of the genetic variation of the species of interest is required. Subsequently, alternative strategies that can be used to improve the quality and yield of A/S are demanded. It has been shown that phytohormones and endophytic bacteria could boost A/S production but the underlying molecular mechanisms remain largely unknown. This thesis aims to characterize genetic variation in two selected A/S producing species of the genus Alkanna and to elucidate pathways and genes associated with enhanced A/S biosynthesis during inoculation of Lithospermum officinale with phytohormones and one of its endophytic bacteria. In chapter one, restriction site-associated DNA sequencing and ploidy-aware genotyping was carried out to investigate the genetic structure in Alkanna tinctoria and Alkanna sieberi, representative A/S producing species of Boraginaceae in Greece. A strong regional genetic structure in 14 analyzed Alkanna tinctoria Greek mainland populations along the north-south gradient together with signals of interspecific admixture influencing genetic variation of Greek mainland and island populations were observed. An additional investigation of Alkanna tinctoria populations showed that both neutral processes and selection have played a role in shaping present day genetic variation in this species. The second chapter aims to provide molecular insight into the regulation of A/S biosynthesis in response to the phytohormones methyl jasmonate and salicylic acid in Lithospermum officinale. Analysis of A/S profiles confirmed that methyl jasmonate but not salicylic acid promotes A/S biosynthesis. By means of comparative transcriptomics, this study further provides evidence for the important role of the precursor mevalonate pathway in phytohormone-mediated A/S production. Integrative analysis via weighted gene co-expression network analysis revealed several structural and regulatory candidates involved in the A/S biosynthetic pathway. The final chapter investigates the possible transcriptional mechanisms involved in the endophytic bacterium Chitinophaga sp. strain R-73072-mediated A/S biosynthesis in Lithospermum officinale. R-73072 significantly enhances A/S production in different Boraginaceae members. Using comparative transcriptomics, it is shown that R-73072 substantially modulated the root transcriptome of Lithospermum officinale, where differentially expressed genes were primarily involved in plant defense, secondary metabolism, and phytohormonal signaling. Higher expression of genes involved in jasmonate and ethylene biosynthesis and signaling further suggest that these phytohormones could be involved in R-73072-mediated A/S biosynthesis. In addition, R-73072 activated not only local defense responses but also induced systemic resistance by reducing fungal infection in foliar tissues.