Periodic Reporting for period 2 - MICROMETABOLITE (“MICROMETABOLITE” – Research Training Network on the Microbial Enhancement of Bioactive Secondary Metabolite Production in Plants)
Reporting period: 2019-05-01 to 2021-10-31
Research in MICROMETABOLITE was focused on medicinal plants and their associated microorganisms. Plant-associated microorganisms such as endophytic bacteria and fungi (which reside inside living plants) and soil-borne, mycorrhizal fungi are essential to optimum plant health and growth. They are physiologically tightly interlinked with their hosts, and, hence, may induce or modulate the production of plant secondary metabolites. Microbial activities that enhance plant nutrient uptake may influence major synthesis pathways, in addition to external influences from herbivores, pathogens, and the abiotic environment. Moreover, endophytic bacteria and fungi themselves are known as producers of various plant-derived bioactive compounds.
In MICROMETABOLITE, we extracted from the roots of Lithospermum and Alkanna plants belonging to the Boraginaceae family various naphtoquinone derivatives (e.g. alkannin and shikonin, A/S). These compounds are renowned for their positive effects in tissue regeneration and wound healing and have proven antimicrobial, anti-inflammatory, anti-HIV-1, and anti-cancer activities. Boraginaceae-derived compounds are already used in various cosmetics and medicinal products, and there is growing interest in expanding the range of pharmaceutical application. However, synthesis in plant cultivation systems is often inconsistent and gives low yields as compared with wild plants growing on their native sites, which puts limits to commercialization. Research work done within the project explored the plant-associated microflora of Lithospermum and Alkanna plants with the aim of utilizing microorganisms for improving the production of bioactive secondary plant metabolites.
In MICROMETABOLITE, we extracted from the roots of Lithospermum and Alkanna plants belonging to the Boraginaceae family various naphtoquinone derivatives (e.g. alkannin and shikonin, A/S). These compounds are renowned for their positive effects in tissue regeneration and wound healing and have proven antimicrobial, anti-inflammatory, anti-HIV-1, and anti-cancer activities. Boraginaceae-derived compounds are already used in various cosmetics and medicinal products, and there is growing interest in expanding the range of pharmaceutical application. However, synthesis in plant cultivation systems is often inconsistent and gives low yields as compared with wild plants growing on their native sites, which puts limits to commercialization. Research work done within the project explored the plant-associated microflora of Lithospermum and Alkanna plants with the aim of utilizing microorganisms for improving the production of bioactive secondary plant metabolites.
We analysed relationships between plant content of A/S or other secondary metabolites, the plant genotype, and the plant-associated microflora. For this, Alkanna tinctoria plants were cultivated in the greenhouse and sampled for analysing and identifying A/S and derivatives. The fruiting stage was found most promising regarding plant A/S production, and it was shown that alkannin derivatives predominated over shikonin.
Further, A. tinctoria iplants were investigated that had been collected from 14 different localities in mainland Greece together with A. sieberi plants collected from two localities on the island of Crete. Following population genetics analysis, the genetic structure of A. tinctoria and the related species A. sieberi was inferred and the main drivers of genetic variation in A. tinctoria were identified. Here, we found a moderate intrapopulation genetic diversity in A. tinctoria, but significant regional differentiation, together with clear evidence of admixture between mainland and island congeners. The genomic variation revealed can be used to further inform and predict the structure of phenotypic variation (e.g. alkannin and shikonin production) in this important medicinal plant.
Genotyping analyses suggested that the plant microbiome composition was influenced by the active recruitment of microbes by the plant from the soil through the root system, besides by host genetic factors and possibly the heritability of the microbiome. The diversity of the microbiome was assessed in samples of wild Echium vulgare from Austria, wild A. tinctoria sampled in different parts of Greece and A. tinctoria species grown under greenhouse conditions on three different soils. Regarding the microbial communities associated with A/S producing plants, Burkholderiaceae, Pseudomonadaceae and Xanthobacteriaceae were the most common bacterial taxax while Plectosphaerella, Penicillium, Fusarium and Trichoderma sp. were the most common fungal taxa.
An in vitro hairy root bioassay was set up using A. tinctoria, where four Agrobacterium strains were shown to increase A/S production in the roots. In vitro experiments with Lithospermum officinale revealed that six bacterial treatments significantly increased shoot length and partly also shoot and/or root dry weight.
Targeted and non-targeted metabolomics analyses showed that three bacterial strains induced a significant increase of A/S and their derivatives in L. officinale and that individual bacterial treatments evoked different effects on the root metabolomeAnalysis of the identified compounds revealed novel regulation patterns of the production of secondary metabolites in L. officinale involving bacteria. Whole genome sequences were determined for 112 bacterial strains, revealing richness in secondary metabolite pathways.
Novel in vitro and in vivo propagation systems of Boraginaceae plants associated with AMF and bacteria were established and associations of both A. officinalis and A. tinctoria with different AMF species were provided in in vivo conditions. Experiments with A. tinctoria plants were performed to study the impact of AMF on alkannin and shikonin (A/S) derivatives production and expression of genes involved in the respective biosynthetic pathways. An AMF species from Greece induced high production of A/S derivatives in A. tinctoria roots.
A Nutrient Film Technique (NFT) cultivation system was developed to provide a non-destructive method for extraction of hydroxynaphthoquinones (A/S derivatives) produced by A. tinctoria plants. In a proof-of-concept experiment, the NFT system was combined with the use of a new eco-friendly category of solvent, i.e. natural deep eutectic solvents (NADESs). Several NADESs were investigated for hydroxynaphtho-quinone extraction efficiencies, and fifteen NADESs components were selected based on their natural origin, environmental-friendliness, and low toxicity, which were the basis for a total of sixty-eight tailor-made solvents. Subsequent screening experiments showed that all the NADESs prepared can be used to extract hydroxynaphthoquinones. Extraction using NADES composed of levulinic acid and glucose was investigated on a larger scale, showing efficient recovery and acceptable robustness, with a promising capacity of the NADES to be recycled. Also, downstream processes were applied for isolating and purifying A/S derivatives from Alkanna tinctoria roots.
Further, A. tinctoria iplants were investigated that had been collected from 14 different localities in mainland Greece together with A. sieberi plants collected from two localities on the island of Crete. Following population genetics analysis, the genetic structure of A. tinctoria and the related species A. sieberi was inferred and the main drivers of genetic variation in A. tinctoria were identified. Here, we found a moderate intrapopulation genetic diversity in A. tinctoria, but significant regional differentiation, together with clear evidence of admixture between mainland and island congeners. The genomic variation revealed can be used to further inform and predict the structure of phenotypic variation (e.g. alkannin and shikonin production) in this important medicinal plant.
Genotyping analyses suggested that the plant microbiome composition was influenced by the active recruitment of microbes by the plant from the soil through the root system, besides by host genetic factors and possibly the heritability of the microbiome. The diversity of the microbiome was assessed in samples of wild Echium vulgare from Austria, wild A. tinctoria sampled in different parts of Greece and A. tinctoria species grown under greenhouse conditions on three different soils. Regarding the microbial communities associated with A/S producing plants, Burkholderiaceae, Pseudomonadaceae and Xanthobacteriaceae were the most common bacterial taxax while Plectosphaerella, Penicillium, Fusarium and Trichoderma sp. were the most common fungal taxa.
An in vitro hairy root bioassay was set up using A. tinctoria, where four Agrobacterium strains were shown to increase A/S production in the roots. In vitro experiments with Lithospermum officinale revealed that six bacterial treatments significantly increased shoot length and partly also shoot and/or root dry weight.
Targeted and non-targeted metabolomics analyses showed that three bacterial strains induced a significant increase of A/S and their derivatives in L. officinale and that individual bacterial treatments evoked different effects on the root metabolomeAnalysis of the identified compounds revealed novel regulation patterns of the production of secondary metabolites in L. officinale involving bacteria. Whole genome sequences were determined for 112 bacterial strains, revealing richness in secondary metabolite pathways.
Novel in vitro and in vivo propagation systems of Boraginaceae plants associated with AMF and bacteria were established and associations of both A. officinalis and A. tinctoria with different AMF species were provided in in vivo conditions. Experiments with A. tinctoria plants were performed to study the impact of AMF on alkannin and shikonin (A/S) derivatives production and expression of genes involved in the respective biosynthetic pathways. An AMF species from Greece induced high production of A/S derivatives in A. tinctoria roots.
A Nutrient Film Technique (NFT) cultivation system was developed to provide a non-destructive method for extraction of hydroxynaphthoquinones (A/S derivatives) produced by A. tinctoria plants. In a proof-of-concept experiment, the NFT system was combined with the use of a new eco-friendly category of solvent, i.e. natural deep eutectic solvents (NADESs). Several NADESs were investigated for hydroxynaphtho-quinone extraction efficiencies, and fifteen NADESs components were selected based on their natural origin, environmental-friendliness, and low toxicity, which were the basis for a total of sixty-eight tailor-made solvents. Subsequent screening experiments showed that all the NADESs prepared can be used to extract hydroxynaphthoquinones. Extraction using NADES composed of levulinic acid and glucose was investigated on a larger scale, showing efficient recovery and acceptable robustness, with a promising capacity of the NADES to be recycled. Also, downstream processes were applied for isolating and purifying A/S derivatives from Alkanna tinctoria roots.
Boraginaceae secondary metabolites were analysed in dependence of the plant genotype, and it was demonstrated that metabolite quantity and quality are influenced by plant-associated microorganisms. This research gave way to establishing a new, innovative plant production system that integrates microorganisms and is operated with a specific protocol. Finally, Alkanna tinctoria extracts were produced at pilot scale. Thus, MICROMETABOLITE’s research provided new knowledge and new processes that may be implemented by industry, which will widen the market’s repertoire of Boraginaceae plant-based pharmaceutics and cosmetics.