CRISPR/Cas9-Mediated Genome Editing in Comfrey (Symphytum officinale) Hairy Roots Results in the Complete Eradication of Pyrrolizidine Alkaloids.

Comfrey (Symphytum officinale) is a medicinal plant with anti-inflammatory, analgesic, and proliferative properties. However, its pharmaceutical application is hampered by the co-occurrence of toxic pyrrolizidine alkaloids (PAs) in its tissues. Using a CRISPR/Cas9-based approach, we introduced detrimental mutations into the hss gene encoding homospermidine synthase (HSS), the first pathway-specific enzyme of PA biosynthesis. The resulting hairy root (HR) lines were analyzed for the type of gene-editing effect that they exhibited and for their homospermidine and PA content. Inactivation of only one of the two hss alleles resulted in HRs with significantly reduced levels of homospermidine and PAs, whereas no alkaloids were detectable in HRs with two inactivated hss alleles. PAs were detectable once again after the HSS-deficient HRs were fed homospermidine confirming that the inability of these roots to produce PAs was only attributable to the inactivated HSS and not to any unidentified off-target effect of the CRISPR/Cas9 approach. Further analyses showed that PA-free HRs possessed, at least in traces, detectable amounts of homospermidine, and that the PA patterns of manipulated HRs were different from those of control lines. These observations are discussed with regard to the potential use of such a CRISPR/Cas9-mediated approach for the economical exploitation of in vitro systems in a medicinal plant and for further studies of PA biosynthesis in non-model plants.

LC-ESI-FT-MSn Metabolite Profiling of Symphytum officinale L. Roots Leads to Isolation of Comfreyn A, an Unusual Arylnaphthalene Lignan.

Preparations of comfrey (Symphytum officinale L.) roots are used topically to reduce inflammation. Comfrey anti-inflammatory and analgesic properties have been proven in clinical studies. However, the bioactive compounds associated with these therapeutic activities are yet to be identified. An LC-ESI-Orbitrap-MSn metabolite profile of a hydroalcoholic extract of comfrey root guided the identification of 20 compounds, including a new arylnaphthalene lignan bearing a rare δ-lactone ring, named comfreyn A. Its structure was determined using extensive 2D NMR and ESI-MS experiments. Additionally, the occurrence of malaxinic acid, caffeic acid ethyl ester, along with the lignans ternifoliuslignan D, 3-carboxy-6,7-dihydroxy-1-(3',4'-dihydroxyphenyl) -naphthalene, globoidnan A and B, and rabdosiin was reported in S. officinale for the first time. These results helped to redefine the metabolite profile of this medicinal plant. Finally, caffeic acid ethyl ester and comfreyn A were found to significantly inhibit E-selectin expression in IL-1ß stimulated human umbilical vein endothelial cells (HUVEC), with EC values of 64 and 50 µM, respectively.

Thermal conversion of a promising phytoremediation plant (Symphytum officinale L.) into biochar: Dynamic of potentially toxic elements and environmental acceptability assessment of the biochar.

Symphytum officinale L., as a hyperaccumulator, was pyrolyzed into biochar at 350, 550, and 750 °C, respectively. PTEs could be enriched in biochars except Cd volatilized greatly at 750 °C. In order to evaluate the environmental acceptability of biochars, a series of sequential and single extractions and biochar oxidation procedures were performed for simulating different environmental conditions. There was a sharp decline in PTEs release under various conditions when the temperature above 550 °C, indicating PTEs might transform into more stable forms at higher temperature. Thus, increasing the pyrolysis temperature is helpful for reducing biochar phytotoxicity, suppressing biochar leaching and improving biochar environmental safety. Moreover, the economic feasibility analysis of the biochar confirmed the practicability of it. Findings from this work illustrated that biochars pyrolyzed from Symphytum officinale L. at the temperature higher than 550 °C might be environmental acceptable, which is beneficial for biochar application.

Specific Distribution of Pyrrolizidine Alkaloids in Floral Parts of Comfrey (Symphytum officinale) and its Implications for Flower Ecology.

Pyrrolizidine alkaloids (PAs) are a typical class of plant secondary metabolites that are constitutively produced as part of the plant's chemical defense. While roots are a well-established site of pyrrolizidine alkaloid biosynthesis, comfrey plants (Symphytum officinale; Boraginaceae) have been shown to additionally activate alkaloid production in specialized leaves and accumulate PAs in flowers during a short developmental stage in inflorescence development. To gain a better understanding of the accumulation and role of PAs in comfrey flowers and fruits, we have dissected and analyzed their tissues for PA content and patterns. PAs are almost exclusively accumulated in the ovaries, while petals, sepals, and pollen hardly contain PAs. High levels of PAs are detectable in the fruit, but the elaiosome was shown to be PA free. The absence of 7-acetyllycopsamine in floral parts while present in leaves and roots suggests that the additional site of PA biosynthesis provides the pool of PAs for translocation to floral structures. Our data suggest that PA accumulation has to be understood as a highly dynamic system resulting from a combination of efficient transport and additional sites of synthesis that are only temporarily active. Our findings are further discussed in the context of the ecological roles of PAs in comfrey flowers.

Identification of a Second Site of Pyrrolizidine Alkaloid Biosynthesis in Comfrey to Boost Plant Defense in Floral Stage.

Pyrrolizidine alkaloids (PAs) are toxic secondary metabolites that are found in several distantly related families of the angiosperms. The first specific step in PA biosynthesis is catalyzed by homospermidine synthase (HSS), which has been recruited several times independently by duplication of the gene encoding deoxyhypusine synthase, an enzyme involved in the posttranslational activation of the eukaryotic initiation factor 5A. HSS shows highly diverse spatiotemporal gene expression in various PA-producing species. In comfrey (Symphytum officinale; Boraginaceae), PAs are reported to be synthesized in the roots, with HSS being localized in cells of the root endodermis. Here, we show that comfrey plants activate a second site of HSS expression when inflorescences start to develop. HSS has been localized in the bundle sheath cells of specific leaves. Tracer feeding experiments have confirmed that these young leaves express not only HSS but the whole PA biosynthetic route. This second site of PA biosynthesis results in drastically increased PA levels within the inflorescences. The boost of PA biosynthesis is proposed to guarantee optimal protection especially of the reproductive structures.

Metabolism, genotoxicity, and carcinogenicity of comfrey.

Comfrey has been consumed by humans as a vegetable and a tea and used as an herbal medicine for more than 2000 years. Comfrey, however, produces hepatotoxicity in livestock and humans and carcinogenicity in experimental animals. Comfrey contains as many as 14 pyrrolizidine alkaloids (PA), including 7-acetylintermedine, 7-acetyllycopsamine, echimidine, intermedine, lasiocarpine, lycopsamine, myoscorpine, symlandine, symphytine, and symviridine. The mechanisms underlying comfrey-induced genotoxicity and carcinogenicity are still not fully understood. The available evidence suggests that the active metabolites of PA in comfrey interact with DNA in liver endothelial cells and hepatocytes, resulting in DNA damage, mutation induction, and cancer development. Genotoxicities attributed to comfrey and riddelliine (a representative genotoxic PA and a proven rodent mutagen and carcinogen) are discussed in this review. Both of these compounds induced similar profiles of 6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP)-derived DNA adducts and similar mutation spectra. Further, the two agents share common mechanisms of drug metabolism and carcinogenesis. Overall, comfrey is mutagenic in liver, and PA contained in comfrey appear to be responsible for comfrey-induced toxicity and tumor induction.

Hydrocarbon footprints as a record of bumblebee flower visitation.

Bumblebees leave traces of cuticular hydrocarbons on flowers they visit, with the amount deposited being positively related to the number of visits. We asked whether such footprint hydrocarbons are retained on flowers for sufficiently long periods of time so as to reflect bee visitation in pollination studies. In laboratory experiments, flower corollae (Primula veris, Digitalis grandiflora) visited by Bombus terrestris workers retained bee-derived nonacosenes (C(29)H(58)) in near-unchanged quantities for 24 hours, both at 15 and 25 degrees C. Additionally, synthetic (Z)-9-tricosene applied to flower corollae of the deadnettle Lamium maculatum was retained for 48 hours in an unchanged quantity. In a field survey, the amount of footprint alkenes on flowers of comfrey (Symphytum officinale) plants was positively correlated with the number of bumblebee visits that those plants had received during the day. Together, these data suggest that flowers retain a long-term quantitative record of bumblebee visitation. The analysis of petal extracts by gas chromatography could provide a cheap and reliable way of quantifying bumblebee visits in landscape scale studies of pollination.

Lead chelation to immobilised Symphytum officinale L. (comfrey) root tannins.

Reported correlations between tannin level and metal accumulation within plant tissues suggest that metal-chelating tannins may help plants to tolerate toxic levels of heavy metal contaminants. This paper supports such correlations using a new method that demonstrated the ability of plant tannins to chelate heavy metals, and showed that the relative levels of tannins in tissues were quantitatively related to lead chelation in vitro. Using this in vitro metal chelation method, we showed that immobilised tannins prepared from lateral roots of Symphytum officinale L., that contained high tannin levels, chelated 3.5 times more lead than those from main roots with lower tannin levels. This trend was confirmed using increasing concentrations of tannins from a single root type, and using purified tannins (tannic acid) from Chinese gallnuts. This study presents a new, simple, and reliable method that demonstrates direct lead-tannin chelation. In relation to phytoremediation, it also suggests that plant roots with more 'built-in' tannins may advantageously accumulate more lead.

Analysis of gene expression changes in relation to toxicity and tumorigenesis in the livers of Big Blue transgenic rats fed comfrey (Symphytum officinale).

BACKGROUND: Comfrey is consumed by humans as a vegetable and a tea, and has been used as an herbal medicine for more than 2000 years. Comfrey, however, is hepatotoxic in livestock and humans and carcinogenic in experimental animals. Our previous study suggested that comfrey induces liver tumors by a genotoxic mechanism and that the pyrrolizidine alkaloids in the plant are responsible for mutation induction and tumor initiation in rat liver. RESULTS: In this study, we identified comfrey-induced gene expression profile in the livers of rats. Groups of 6 male transgenic Big Blue rats were fed a basal diet and a diet containing 8% comfrey roots, a dose that resulted in liver tumors in a previous carcinogenicity bioassay. The animals were treated for 12 weeks and sacrificed one day after the final treatment. We used a rat microarray containing 26,857 genes to perform genome-wide gene expression studies. Dietary comfrey resulted in marked changes in liver gene expression, as well as in significant decreases in the body weight and increases in liver mutant frequency. When a two-fold cutoff value and a P-value less than 0.01 were selected, 2,726 genes were identified as differentially expressed in comfrey-fed rats compared to control animals. Among these genes, there were 1,617 genes associated by Ingenuity Pathway Analysis with particular functions, and the differentially expressed genes in comfrey-fed rat livers were involved in metabolism, injury of endothelial cells, and liver injury and abnormalities, including liver fibrosis and cancer development. CONCLUSION: The gene expression profile provides us a better understanding of underlying mechanisms for comfrey-induced hepatic toxicity. Integration of gene expression changes with known pathological changes can be used to formulate a mechanistic scheme for comfrey-induced liver toxicity and tumorigenesis.