Clock protein LHY targets SNAT1 and negatively regulates the biosynthesis of melatonin in Hypericum perforatum.

Hypericum perforatum, also known as "natural fluoxetine," is a commonly used herbal remedy for treating depression. It is unclear whether melatonin in plants regulated by the endogenous circadian clock system is like in vertebrates. In this work, we found that the melatonin signal and melatonin biosynthesis gene, serotonin N-acetyltransferase HpSNAT1, oscillates in a 24-hour cycle in H. perforatum. First, we constructed a yeast complementary DNA library of H. perforatum and found a clock protein HpLHY that can directly bind to the HpSNAT1 promoter. Second, it was confirmed that HpLHY inhibits the expression of HpSNAT1 by targeting the Evening Element. Last, it indicated that HpLHY-overexpressing plants had reduced levels of melatonin in 12-hour light/12-hour dark cycle photoperiod, while loss-of-function mutants exhibited high levels, but this rhythm seems to disappear as well. The results revealed the regulatory role of LHY in melatonin biosynthesis, which may make an important contribution to the field of melatonin synthesis regulation.

Single-cell RNA sequencing facilitates the elucidation of the complete biosynthesis of the antidepressant hyperforin in St. John's wort.

Hyperforin is the compound responsible for the effectiveness of St. John's wort (Hypericum perforatum) as an antidepressant, but its complete biosynthetic pathway remains unknown. Gene discovery based on co-expression analysis of bulk RNA-sequencing data or genome mining failed to discover the missing steps in hyperforin biosynthesis. In this study, we sequenced the 1.54-Gb tetraploid H. perforatum genome assembled into 32 chromosomes with the scaffold N50 value of 42.44 Mb. By single-cell RNA sequencing, we identified a type of cell, "Hyper cells", wherein hyperforin biosynthesis de novo takes place in both the leaves and flowers. Through pathway reconstitution in yeast and tobacco, we identified and characterized four transmembrane prenyltransferases (HpPT1-4) that are localized at the plastid envelope and complete the hyperforin biosynthetic pathway. The hyperforin polycyclic scaffold is created by a reaction cascade involving an irregular isoprenoid coupling and a tandem cyclization. Our findings reveal how and where hyperforin is biosynthesized, enabling synthetic-biology reconstitution of the complete pathway. Thus, this study not only deepens our comprehension of specialized metabolism at the cellular level but also provides strategic guidance for elucidation of the biosynthetic pathways of other specializied metabolites in plants.

Phytochemical Analysis, Biological Activities, and Docking of Phenolics from Shoot Cultures of Hypericum perforatum L. Transformed by Agrobacterium rhizogenes.

Hypericum perforatum transformed shoot lines (TSL) regenerated from corresponding hairy roots and non-transformed shoots (NTS) were comparatively evaluated for their phenolic compound contents and in vitro inhibitory capacity against target enzymes (monoamine oxidase-A, cholinesterases, tyrosinase, α-amylase, α-glucosidase, lipase, and cholesterol esterase). Molecular docking was conducted to assess the contribution of dominant phenolic compounds to the enzyme-inhibitory properties of TSL samples. The TSL extracts represent a rich source of chlorogenic acid, epicatechin and procyanidins, quercetin aglycone and glycosides, anthocyanins, naphthodianthrones, acyl-phloroglucinols, and xanthones. Concerning in vitro bioactivity assays, TSL displayed significantly higher acetylcholinesterase, tyrosinase, α-amylase, pancreatic lipase, and cholesterol esterase inhibitory properties compared to NTS, implying their neuroprotective, antidiabetic, and antiobesity potential. The docking data revealed that pseudohypericin, hyperforin, cadensin G, epicatechin, and chlorogenic acid are superior inhibitors of selected enzymes, exhibiting the lowest binding energy of ligand-receptor complexes. Present data indicate that H. perforatum transformed shoots might be recognized as an excellent biotechnological system for producing phenolic compounds with multiple health benefits.

Differentiated response of Hypericum perforatum to foliar application of selected metabolic modulators: Elicitation potential of chitosan, selenium, and salicylic acid mediated by redox imbalance.

Plants plastically alter their metabolism in response to environmental stimuli, which induces changes in the accumulation of specialized metabolites. This ability can be utilized to manipulate plant phytochemistry in a desired direction. However, the abundance of secondary metabolites in the different plant species, especially medicinal, is enormous; therefore, it is difficult to establish a clear direction for the effects of metabolic modulators on phytochemical composition, especially given the possibility of using different types thereof. In order to gain insight into these changes, we investigated the effects of foliar-applied chitosan (ChL, 100 mg/L), selenium (Se, 10 mg/L), salicylic acid (SA, 150 mg/L), or an equal volume mixture thereof on Hypericum perforatum L. metabolism. Selenium and SA proved to be the more effective than ChL in enhancing the accumulation of phenolic compounds. The greatest increase was found in the concentration of neochlorogenic acid after Se-spraying. The treatment with the elicitors generally increased the concentration of identified flavonoids, but not the level of naphthodianthrone or phloroglucinol metabolites. The most pronounced response was observed on day 10 following the application of the compounds, and is likely the consequence of elevated levels of O2-˙, free proline, and modulated activity of enzymatic antioxidants.

In silico prediction of polyketide biosynthetic gene clusters in the genomes of Hypericum-borne endophytic fungi.

BACKGROUND: The search for new bioactive natural compounds with anticancer activity is still of great importance. Even though their potential for diagnostics and treatment of cancer has already been proved, the availability is still limited. Hypericin, a naphthodianthrone isolated essentially from plant source Hypericum perforatum L. along with other related anthraquinones and bisanthraquinones belongs to this group of compounds. Although it has been proven that hypericin is synthesized by the polyketide pathway in plants, none of the candidate genes coding for key enzymes has been experimentally validated yet. Despite the rare occurrence of anthraquinones in plants, their presence in microorganisms, including endophytic fungi, is quite common. Unlike plants, several biosynthetic genes grouped into clusters (BGCs) in fungal endophytes have already been characterized. RESULTS: The aim of this work was to predict, identify and characterize the anthraquinone BGCs in de novo assembled and functionally annotated genomes of selected endophytic fungal isolates (Fusarium oxysporum, Plectosphaerella cucumerina, Scedosporium apiospermum, Diaporthe eres, Canariomyces subthermophilus) obtained from different tissues of Hypericum spp. The number of predicted type I polyketide synthase (PKS) BGCs in the studied genomes varied. The non-reducing type I PKS lacking thioesterase domain and adjacent discrete gene encoding protein with product release function were identified only in the genomes of C. subthermophilus and D. eres. A candidate bisanthraquinone BGC was predicted in C. subthermophilus genome and comprised genes coding the enzymes that catalyze formation of the basic anthraquinone skeleton (PKS, metallo-beta-lactamase, decarboxylase, anthrone oxygenase), putative dimerization enzyme (cytochrome P450 monooxygenase), other tailoring enzymes (oxidoreductase, dehydrogenase/reductase), and non-catalytic proteins (fungal transcription factor, transporter protein). CONCLUSIONS: The results provide an insight into genetic background of anthraquinone biosynthesis in Hypericum-borne endophytes. The predicted bisanthraquinone gene cluster represents a basis for functional validation of the candidate biosynthetic genes in a simple eukaryotic system as a prospective biotechnological alternative for production of hypericin and related bioactive anthraquinones.

Regiodivergent biosynthesis of bridged bicyclononanes.

Medicinal compounds from plants include bicyclo[3.3.1]nonane derivatives, the majority of which are polycyclic polyprenylated acylphloroglucinols (PPAPs). Prototype molecules are hyperforin, the antidepressant constituent of St. John's wort, and garcinol, a potential anticancer compound. Their complex structures have inspired innovative chemical syntheses, however, their biosynthesis in plants is still enigmatic. PPAPs are divided into two subclasses, named type A and B. Here we identify both types in Hypericum sampsonii plants and isolate two enzymes that regiodivergently convert a common precursor to pivotal type A and B products. Molecular modelling and substrate docking studies reveal inverted substrate binding modes in the two active site cavities. We identify amino acids that stabilize these alternative binding scenarios and use reciprocal mutagenesis to interconvert the enzymatic activities. Our studies elucidate the unique biochemistry that yields type A and B bicyclo[3.3.1]nonane cores in plants, thereby providing key building blocks for biotechnological efforts to sustainably produce these complex compounds for preclinical development.

Do nanoparticles delivered to roots affect plant secondary metabolism? A comprehensive analysis in float seedling cultures of Hypericum perforatum L.

Since nanoparticles (NPs) released into the environment from household or industrial wastes and applied directly on plants as agrochemicals can accumulate in the rhizosphere, it is imperative to understand how these NPs affect plant secondary metabolism upon their contact with the roots of intact plants. Here, the effects of Pd, Au, ZnO and Fe2O3 NPs on secondary metabolism were comprehensively investigated in Hypericum perforatum L float seedlings by analyzing 41 major secondary metabolites using ultra-performance liquid chromatography coupled with photodiode array, fluorescence detector and high-resolution mass spectrometry (UPLC-PDA-FLR-HRMS). The results showed that exposure of H. perforatum roots to Pd, Au, ZnO and Fe2O3 NPs rapidly led to fluctuations in the levels of secondary metabolites. Although these fluctuations did not correlate with NP type, concentration and duration of treatment, a total of 22 compounds were significantly altered by the NPs tested. In particular, 1 ppm Au increased the content of quercetin 3-(2″-acetylgalactoside), cadensin G and leutoskyrin by 5.02-, 2.12- and 2.58-fold, respectively after 24 h; 25 ppm Pd NPs led to a 2.1-fold increase in miquelianin content after 6 h; 50 ppm Fe2O3 NPs increased the level of furohyperforin by 3.09-fold and decreased the content of miquelianin 5.22-fold after 24 h and 50 ppm ZnO led to a 2.13-fold increase in hypericin after 48 h. These results emphasise the need to understand the intricate interplay between NPs and plant secondary metabolism in order to enable safer and efficient applications of NPs in agriculture.

Effect of Tacrolimus Formulation (Prolonged-Release vs Immediate-Release) on Its Susceptibility to Drug-Drug Interactions with St. John's Wort.

Tacrolimus is metabolized by cytochrome P450 3A (CYP3A) and is susceptible to interactions with the CYP3A and P-glycoprotein inducer St. John's Wort (SJW). CYP3A isozymes are predominantly expressed in the small intestine and liver. Prolonged-release tacrolimus (PR-Tac) is largely absorbed in distal intestinal segments and is less susceptible to CYP3A inhibition. The effect of induction by SJW is unknown. In this randomized, crossover trial, 18 healthy volunteers received single oral tacrolimus doses (immediate-release [IR]-Tac or PR-Tac, 5 mg each) alone and during induction by SJW. Concentrations were quantified using ultra-high performance liquid chromatography coupled with tandem mass spectrometry and non-compartmental pharmacokinetics were evaluated. SJW decreased IR-Tac exposure (area under the concentration-time curve) to 73% (95% confidence interval 60%-88%) and maximum concentration (Cmax ) to 61% (52%-73%), and PR-Tac exposure to 67% (55%-81%) and Cmax to 69% (58%-82%), with no statistical difference between the 2 formulations. The extent of interaction appeared to be less pronounced in volunteers with higher baseline CYP3A4 activity and in CYP3A5 expressors. In contrast to CYP3A inhibition, CYP3A induction by SJW showed a similar extent of interaction with both tacrolimus formulations. A higher metabolic baseline capacity appeared to attenuate the extent of induction by SJW.

St. John's Wort Formulations Induce Rat CYP3A23-3A1 Independent of Their Hyperforin Content.

The pregnane X receptor (PXR) is a ligand-activated regulator of cytochrome P450 (CYP)3A enzymes. Among the ligands of human PXR is hyperforin, a constituent of St John's wort (SJW) extracts and potent inducer of human CYP3A4. It was the aim of this study to compare the effect of hyperforin and SJW formulations controlled for its content on CYP3A23-3A1 in rats. Hyperiplant was used as it contains a high hyperforin content and Rebalance because it is controlled for a low hyperforin content. In silico analysis revealed a weak hyperforin-rPXR binding affinity, which was further supported in cell-based reporter gene assays showing no hyperforin-mediated reporter activation in presence of rPXR. However, cellular exposure to Hyperiplant and Rebalance transactivated the CYP3A reporter 3.8-fold and 2.8-fold, respectively, and they induced Cyp3a23-3a1 mRNA expression in rat hepatoma cells compared with control 48-fold and 18-fold, respectively. In Wistar rats treated for 10 days with 400 mg/kg of Hyperiplant, we observed 1.8 times the Cyp3a23-3a1 mRNA expression, a 2.6-fold higher CYP3A23-3A1 protein amount, and a 1.6-fold increase in activity compared with controls. For Rebalance we only observed a 1.8-fold hepatic increase of CYP3A23-3A1 protein compared with control animals. Even though there are differing effects on rCyp3a23-3a1/CYP3A23-3A1 in rat liver reflecting the hyperforin content of the SJW extracts, the modulation is most likely not linked to an interaction of hyperforin with rPXR. SIGNIFICANCE STATEMENT: Treatment with St John's wort (SJW) has been reported to affect CYP3A expression and activity in rats. Our comparative study further supports this finding but shows that the pregnane X receptor-ligand hyperforin is not the driving force for changes in rat CYP3A23-3A1 expression and function in vivo and in vitro. Importantly, CYP3A induction mimics findings in humans, but our results suggest that another so far unknown constituent of SJW is responsible for the expression- and function-modifying effects in rat liver.

Comparative Genomics and Physiological Investigations Supported Multifaceted Plant Growth-Promoting Activities in Two Hypericum perforatum L.-Associated Plant Growth-Promoting Rhizobacteria for Microbe-Assisted Cultivation.

Plants are no longer considered standalone entities; instead, they harbor a diverse community of plant growth-promoting rhizobacteria (PGPR) that aid them in nutrient acquisition and can also deliver resilience. Host plants recognize PGPR in a strain-specific manner; therefore, introducing untargeted PGPR might produce unsatisfactory crop yields. Consequently, to develop a microbe-assisted Hypericum perforatum L. cultivation technique, 31 rhizobacteria were isolated from the plant's high-altitude Indian western Himalayan natural habitat and in vitro characterized for multiple plant growth-promoting attributes. Among 31 rhizobacterial isolates, 26 produced 0.59 to 85.29 µg mL-1 indole-3-acetic acid and solubilized 15.77 to 71.43 µg mL-1 inorganic phosphate; 21 produced 63.12 to 99.92% siderophore units, and 15 exhibited 103.60 to 1,296.42 nmol α-ketobutyrate mg-1 protein h-1 1-aminocyclopropane-1-carboxylate deaminase (ACCD) activity. Based on superior plant growth-promoting attributes, eight statistically significant multifarious PGPR were further evaluated for an in planta plant growth-promotion assay under poly greenhouse conditions. Plants treated with Kosakonia cowanii HypNH10 and Rahnella variigena HypNH18 showed, by significant amounts, the highest photosynthetic pigments and performance, eventually leading to the highest biomass accumulation. Comparative genome analysis and comprehensive genome mining unraveled their unique genetic features, such as adaptation to the host plant's immune system and specialized metabolites. Moreover, the strains harbor several functional genes regulating direct and indirect plant growth-promotion mechanisms through nutrient acquisition, phytohormone production, and stress alleviation. In essence, the current study endorsed strains HypNH10 and HypNH18 as cogent candidates for microbe-assisted H. perforatum cultivation by highlighting their exclusive genomic signatures, which suggest their unison, compatibility, and multifaceted beneficial interactions with their host and support the excellent plant growth-promotion performance observed in the greenhouse trial. IMPORTANCE Hypericum perforatum L. (St. John's wort) herbal preparations are among the top-selling products to treat depression worldwide. A significant portion of the overall Hypericum supply is sourced through wild collection, prompting a rapid decline in their natural stands. Crop cultivation seems lucrative, although cultivable land and its existing rhizomicrobiome are well suited for traditional crops, and its sudden introduction can create soil microbiome dysbiosis. Also, the conventional plant domestication procedures with increased reliance on agrochemicals can reduce the diversity of the associated rhizomicrobiome and plants' ability to interact with plant growth-promoting microorganisms, leading to unsatisfactory crop production alongside harmful environmental effects. Cultivating H. perforatum with crop-associated beneficial rhizobacteria can reconcile such concerns. Based on a combinatorial in vitro, in vivo plant growth-promotion assay and in silico prediction of plant growth-promoting traits, here we recommend two H. perforatum-associated PGPR, Kosakonia cowanii HypNH10 and Rahnella variigena HypNH18, to extrapolate as functional bioinoculants for H. perforatum sustainable cultivation.