Dynamics of alkannin/shikonin biosynthesis in response to jasmonate and salicylic acid in Lithospermum officinale.

Alkannin/shikonin and their derivatives are specialised metabolites of high pharmaceutical and ecological importance exclusively produced in the periderm of members of the plant family Boraginaceae. Previous studies have shown that their biosynthesis is induced in response to methyl jasmonate but not salicylic acid, two phytohormones that play important roles in plant defence. However, mechanistic understanding of induction and non-induction remains largely unknown. In the present study, we generated the first comprehensive transcriptomic dataset and metabolite profiles of Lithospermum officinale plants treated with methyl jasmonate and salicylic acid to shed light on the underlying mechanisms. Our results highlight the diverse biological processes activated by both phytohormones and reveal the important regulatory role of the mevalonate pathway in alkannin/shikonin biosynthesis in L. officinale. Furthermore, by modelling a coexpression network, we uncovered structural and novel regulatory candidate genes connected to alkannin/shikonin biosynthesis. Besides providing new mechanistic insights into alkannin/shikonin biosynthesis, the generated methyl jasmonate and salicylic acid elicited expression profiles together with the coexpression networks serve as important functional genomic resources for the scientific community aiming at deepening the understanding of alkannin/shikonin biosynthesis.

Root-associated bacteria modulate the specialised metabolome of Lithospermum officinale L.

Bacteria influence plant growth and development and therefore are attractive resources for applications in agriculture. However, little is known about the impact of these microorganisms on secondary metabolite (SM) production by medicinal plants. Here we assessed, for the first time, the effects of bacteria on the modulation of SM production in the medicinal plant Lithospermum officinale (Boraginaceae family) with a focus on the naphthoquinones alkannin/shikonin and their derivatives (A/Sd). The study was conducted in an in vitro cultivation system developed for that purpose, as well as in a greenhouse. Targeted and non-targeted metabolomics were performed, and expression of the gene PGT encoding for a key enzyme in the A/S biosynthesis pathway was evaluated with qPCR. Three strains, Chitinophaga sp. R-73072, Xanthomonas sp. R-73098 and Pseudomonas sp. R-71838 induced a significant increase of A/Sd in L. officinale in both systems, demonstrating the strength of our approach for screening A/Sd-inducing bacteria. The bacterial treatments altered other plant metabolites derived from the shikimate pathway as well. Our results demonstrate that bacteria influence the biosynthesis of A/Sd and interact with different metabolic pathways. This work highlights the potential of bacteria to increase the production of SM in medicinal plants and reveals new patterns in the metabolome regulation of L. officinale.

Transcriptional dynamics of Chitinophaga sp. strain R-73072-mediated alkannin/shikonin biosynthesis in Lithospermum officinale.

Plants are colonized by a wide range of bacteria, several of which are known to confer benefits to their hosts such as enhancing plant growth and the biosynthesis of secondary metabolites (SMs). Recently, it has been shown that Chitinophaga sp. strain R-73072 enhances the production of alkannin/shikonin, SMs of pharmaceutical and ecological importance. However, the mechanisms by which this bacterial strain increases these SMs in plants are not yet understood. To gain insight into these mechanisms, we analyzed the molecular responses of Lithospermum officinale, an alkannin/shikonin producing member of Boraginaceae, to inoculation with R-73072 in a gnotobiotic system using comparative transcriptomics and targeted metabolite profiling of root samples. We found that R-73072 modulated the expression of 1,328 genes, of which the majority appeared to be involved in plant defense and SMs biosynthesis including alkannin/shikonin derivatives. Importantly, bacterial inoculation induced the expression of genes that predominately participate in jasmonate and ethylene biosynthesis and signaling, suggesting an important role of these phytohormones in R-73072-mediated alkannin/shikonin biosynthesis. A detached leaf bioassay further showed that R-73072 confers systemic protection against Botrytis cinerea. Finally, R-73072-mediated coregulation of genes involved in plant defense and the enhanced production of alkannin/shikonin esters further suggest that these SMs could be important components of the plant defense machinery in alkannin/shikonin producing species.