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

Abstract

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

“…However, R-73072 appeared non-pathogenic to L. officinale as we did not observe any visible symptoms or growth inhibition ( Supplementary Figure 2 ). These findings are in accordance with earlier observations where no growth defects nor a loss in biomass were found during greenhouse and in vitro investigations ( Varela-Alonso et al, 2022 ). Instead, defense-related transcriptional responses upon R-73072 interaction might confer protection against further potentially unwanted microorganisms.…”

“…Recently, Fazal et al (2021) and Rat et al (2021) demonstrated that A/S producing Boraginaceae (e.g., Alkanna tinctoria ; Ahmad et al, 2021 ) host diverse microbial communities, among which a number of cultivable bacteria were shown to improve A/S production in a hairy root culture system. These findings were further supported by a study from Varela-Alonso et al (2022) , in which endophytic bacteria isolated from A. tinctoria also improved A/S biosynthesis in L. officinale , a Boraginaceae widely distributed in Europe and Asia ( Al-Snafi, 2019 ). Among the tested bacterial isolates, Chitinophaga sp.…”

“…Lithospermum officinale in vitro plants (clone 16) were provided by INoQ GmbH (Schnega, Germany) and grown and maintained on a modified Murashige and Skoog (MS mod ) medium, as described in Varela-Alonso et al (2022) . A pure culture of Chitinophaga sp.…”

“…strain R-73072 ( Rat et al, 2021 ) was provided by BCCM/LMG Bacteria Collection (Ghent, Belgium). Bacterial inoculum was prepared as described in Varela-Alonso et al (2022) . Briefly, the bacterium was grown in a 35 ml liquid R2B medium ( Supplementary Data S0A ) at 25°C and 100 rpm for 72 h. A total of 5 ml was pipetted and transferred to a 15 ml tube for enumeration without affecting the rest of the culture.…”

“…Four-week-old plants were selected, and 2–3 cm long shoot cuttings were cut starting from the tip. The shoot cuttings were immediately transferred to the modified Strullu-Romand medium (MSR mod ; Varela-Alonso et al, 2022 ) in contact with the bacterial or PBS suspension. Three shoot cuttings were transferred per glass jar.…”