Exogenous Signaling Molecules Research Articles

Effects of exogenous signaling molecules on anaerobic sludge digestion: Quorum sensing and antibiotic resistance genes

Regulating quorum sensing (QS) signaling molecules could improve wastewater treatment but might increase antibiotic resistance. This study investigated the effects of exogenous C6-HSL on anaerobic sludge under oxytetracycline stress, with a focus on antibiotic resistance genes (ARGs) and the QS response. The results revealed that exogenous oxytetracycline increased the copy number of ARGs by more than 68.8 %. It also facilitated a 3.04-fold increase in the concentration of signaling molecules and increased the abundance of QS genes. Further addition of the C6-HSL accelerated oxytetracycline degradation, and reduced its residual concentration by 70.9 %, alleviating oxytetracycline stress on microbial communities, and correspondingly reducing stress release from AHL by 75.4 %. Importantly, this did not exacerbate antibiotic resistance, with no significant difference (p > 0.05) in the ARG abundance. These findings may provide valuable insights into the relationship between QS process and antibiotic resistance.

The Differentiation Fate of Granulosa Cells and the Regulatory Mechanism in Ovary.

Granulosa cells (GCs) are important drives of the reproductive process, not only the supporting cells for nutrition, but also cells with endocrine functions. Their differentiation and development parallel the entire menstruation period and even during pregnancy, making it tightly linked to the fate of the follicle. To elucidate the underlying mechanism is of great significance for related researches. The life course of GCs is briefly divided into five stages, from epithelial cells to pre-granulosa cells, GCs, mural and cumulus cells, lutein cells, and eventually disappear. A wide variety of genes and transcription factors participate in the regulation of different stages, and more importantly, various hormones secreted by the pituitary gland and GCs themselves play a leading role. These endogenous and exogenous signalling molecules interact to form a cross-linked communication network, promoting the development of GCs. Together with oocytes, theca cells and other functional cells in the ovary, GCs drive one of the most vital biological processes in women.

Dopamine, an exogenous quorum sensing signaling molecule or a modulating factor in Pseudomonas aeruginosa?

Pseudomonas aeruginosa is recognized globally as an opportunistic pathogen of considerable concern due to its high virulence and pathogenicity, especially in immunocompromised individuals. While research has identified several endogenous quorum sensing (QS) signaling molecules that enhance the virulence and pathogenicity of P. aeruginosa, investigations on exogenous QS signaling molecules or modulating factors remain limited. This study found that dopamine serves as an exogenous QS signaling molecule or modulating factor of P. aeruginosa PAO1, enhancing the production of virulence factors and biofilms. Compared to the control group, treatment with 40 μM dopamine resulted in a 33.1 % increase in biofilm formation, 68.1 % increase in swimming mobility, 63.1 % increase in swarming mobility, 147.2 % increase in the signaling molecule 3-oxo-C12-HSL, and 50.5 %, 28.5 %, 27.0 %, and 33.2 % increases in the virulence factors alginate, rhamnolipids, protease, and pyocyanin, respectively. This study further explored the mechanism of dopamine regulating the biofilm formation and virulence of P. aeruginosa PAO1 through transcriptome and metabolome. Transcriptomic analysis showed that dopamine promoted the expression of virulence genes psl, alg, lasA, rhlABC, rml, and phz in P. aeruginosa PAO1. Metabolomic analysis revealed changes in the concentrations of tryptophan, pyruvate, ethanolamine, glycine, 3-hydroxybutyric acid, and alizarin. Furthermore, KEGG enrichment analysis of altered genes and metabolites indicated that dopamine enhanced phenylalanine, tyrosine, and tryptophan in P. aeruginosa PAO1. The results of this study will contribute to the development of novel exogenous QS signaling molecules or modulating factors and advance our understanding of the interactions between P. aeruginosa and the host environment.

Role and mechanism of endoplasmic reticulum stress and NLRP3 inflammasome in acute kidney injury.

Acute kidney injury (AKI) is a common critical condition in clinical practice, characterized by a rapid decline in renal function within a short period. The pathogenesis of AKI is complex and has not been fully elucidated. In recent years, studies have found that the activation of endoplasmic reticulum stress (ERS) and the Nod-like receptor family pyrin domain containing 3 (NLRP3) inflammasome are closely related to the occurrence of AKI. When the kidneys is damaged, the internal environment of the kidney cells is disrupted, leading to the activation of ERS. Excessive ERS can induce apoptosis of renal cells, leading to the occurrence of AKI. Additionally, the NLRP3 inflammasome can mediate the recognition of endogenous and exogenous danger signal molecules by the host, subsequently activating caspase-1, pro-inflammatory cytokines such as IL-1β and IL-18, inducing inflammatory responses, and promoting apoptosis of renal cells. In animal models of AKI, the upregulation of ERS markers is often accompanied by increased expression levels of NLRP3 inflammasome-related proteins, indicating that ERS can regulate the activation process of the NLRP3 inflammasome. Clarifying the role and mechanism of ERS and NLRP3 inflammasome in AKI is expected to provide new insights for the prevention and treatment of AKI.

Chromatin reprogramming and bone regeneration in vitro and in vivo via the microtopography-induced constriction of cell nuclei.

Topographical cues on cells can, through contact guidance, alter cellular plasticity and accelerate the regeneration of cultured tissue. Here we show how changes in the nuclear and cellular morphologies of human mesenchymal stromal cells induced by micropillar patterns via contact guidance influence the conformation of the cells' chromatin and their osteogenic differentiation in vitro and in vivo. The micropillars impacted nuclear architecture, lamin A/C multimerization and 3D chromatin conformation, and the ensuing transcriptional reprogramming enhanced the cells' responsiveness to osteogenic differentiation factors and decreased their plasticity and off-target differentiation. In mice with critical-size cranial defects, implants with micropillar patterns inducing nuclear constriction altered the cells' chromatin conformation and enhanced bone regeneration without the need for exogenous signalling molecules. Our findings suggest that medical device topographies could be designed to facilitate bone regeneration via chromatin reprogramming.

A new strategy for the enrichment of ammonia-oxidizing archaea in wastewater treatment systems: The positive role of quorum-sensing signaling molecules

Ammonia-oxidizing archaea (AOA) play an important role in natural nitrogen cycle, but are difficult to be enriched in wastewater treatment systems. In this experiment, under ambient temperature and high dissolved oxygen, different types of acyl-homoserine lactones (C6-HSL, C8-HSL, C10-HSL, C14-HSL and 3-oxo-C14-HSL) were added to five wastewater nitrification systems to achieve AOA enrichment. Results showed that AOA couldn't be detected in the blank group without the addition of signaling molecules, while the AOA could be detected in all the reactors with the addition. The enrichment effect of AOA was not obvious with added 100 or 200 nmol/L signaling molecules, while the enrichment effect was both obvious with added C8-HSL of 400 nmol/L and C10-HSL of 800 nmol/L. And relative abundance of AOA increased from undetected in the control group to 1.10 % and 0.96 %, respectively. The exogenous signaling molecules may provide new view for AOA enrichment in wastewater treatment systems.

Crucial Triad in Pulp-Dentin Complex Regeneration: Dental Stem Cells, Scaffolds, and Signaling Molecules

BACKGROUND: Pulp damage can lead to dentinogenesis impairment, irreversible pulpitis, or pulp necrosis. Despite being the most used endodontic procedure to treat damaged pulp, root canal therapy only results in nonvital teeth which are prone to fractures and secondary infection. Pulp-dentin regeneration has a potential to regenerate structure similar to normal pulp-dentin complex, and can be achieved by combining dental stem cells, scaffold, and signaling molecules. This article reviews the role of various types of dental stem cells, scaffolds, signaling molecules, and their combinations in regenerating pulp-dentin complex.CONTENT: Dental pulp stem cell (DPSC), stem cell from human exfoliated deciduous teeth (SHED), and dental follicle stem cell (DFSC) were reported to regenerate pulp-dentin complex in situ. SHED might be more promising than DPSCs and DFSCs for regenerating pulp-dentin complex, since SHED have a higher proliferation potential and higher expression levels of signaling molecules. Scaffolds have characteristics resembling extracellular matrix, thus providing a suitable microenvironment for transplanted dental stem cells. To accelerate the regeneration process, exogenous signaling molecules are often delivered together with dental stem cells. Scaffolds and signaling molecules have different regenerative potential, including induction of cell proliferation and migration, formation of pulp- and/or dentin-like tissue, as well as angiogenesis and neurogenesis promotion.SUMMARY: Combinations of dental stem cells, scaffold, and signaling molecules are important to achieve the functional pulp-dentin complex formation. Current trends and future directions on regenerative endodontics should be explored. The right combination of dental stem cells, scaffold, and signaling molecules could be determined based on the patients’ characteristics. Incomplete pulp-dentin regeneration could be overcome by applying dental stem cells, scaffold, and/or signaling molecules in multiple visits.KEYWORDS: pulp-dentin regeneration, regenerative endodontics, dental stem cells, scaffold, signaling molecules

Synergistic interplay between Azospirillum brasilense and exogenous signaling molecule H2S promotes Cd stress resistance and growth in pak choi (Brassica chinensis L.).

Inoculation with growth-promoting rhizobacteria inoculation and the addition of exogenous signaling molecules are two distinct strategies for improving heavy metal resistance and promoting growth in crops through several mechanisms. However, whether rhizobacteria and phyllosphere signaling molecules can act synergistically alleviate heavy metal stress and promote growth and the mechanisms underlying these effects remain unclear. Here, a novel strategy involving the co-application of growth-promoting rhizobacteria and an exogenous signaling molecule was developed to reduce cadmium (Cd) phytotoxicity and promote pak choi growth in Cd-contaminated soil. We found that the co-application of Azospirillum brasilense and hydrogen sulfide (H2S) resulted in significant improvements in shoot biomass and antioxidant enzyme content and a decline in the levels of Cd translocation factors. In addition, this co-application significantly improved pak choi Cd resistance. Furthermore, we observed a significant negative correlation between abscisic acid concentration and Cd content of pak choi and a positive correlation between H2S concentration and biomass. These findings revealed that the co-application of rhizobacteria and exogenous signaling molecules synergistically promoted the growth of vegetable crops subjected to heavy metal stress. Our results may serve as a guide for improving the food safety of crops grown in soil contaminated with heavy metals.

Facilely self-assembled and dual-molecule calibration aptasensor based on SERS for ultra-sensitive detection of tetrodotoxin in pufferfish

Tetrodotoxin (TTX) is one of the most lethal neurotoxins, so the reliable quantitative analysis of TTX is crucial for food and environmental safety monitoring. Herein, a novel dual-molecule calibration aptasensor was developed for detection of TTX based on Surface-enhanced Raman scattering (SERS). The adaptive surface has high affinity recognition sites for the target of interest, which ensures the high specificity and stability of the aptasensor. In addition, the uniquely labeled signal molecules located in the Raman silent region (1800–2400 cm−1) can avoid the interference of other exogenous biological signal molecules. Meanwhile, in quantitative analysis, the SERS signal generated by the reporter is calibrate in real time using the second-order peak of silicon molecule (Si). The detection linear range of the aptasensor was 0.0319 ng/mL-319.27 ng/mL, with a limit of detection (LOD) of 0.024 ng/mL and the excellent uniformity (RSD = 8.8%) for TTX detection. As a promising and versatile detection candidate, the ultra-sensitive quantitative detection aptasensor of TTX had important practical significance, which can offer more favorable persuasion for TTX analysis in real seafood samples.

Mechanism and regulation of filamentous algal-bacterial symbiosis based on microbiological quorum sensing.

In algae-bacteria symbiotic systems, algae and bacteria work cooperatively to reduce aeration demand and carbon emissions during wastewater treatment. However, controlling and stabilizing microbial communities in the conventional algae-bacteria symbiotic systems are complicated and unstable. We, therefore, developed a novel sewage treatment system based on a filamentous algae-bacterial symbiotic granule system (AB) in a photo-sequencing batch reactor (PSBR). The exogenous signal molecules (i.e., N-acyl-homoserine lactones [AHLs]) were applied to promote the algae-bacteria consortia formation. The characteristics and performance of the mature algae-bacterial granular consortia were investigated and then compared with that of the activated sludge (AS) system under identical operating conditions. The chemical oxidation demand (COD) removal efficiencies were greater than 90% in the dynamic synergistic investigation, even without aeration. However, the addition of AHLs (5, 50, and 200 nmol/L) had a negligible effect on treatment performance. In static conditions, 10-9nmol/L of AHLs can significantly regulate the algae-bacteria symbiotic system, and AHLs (<50 nmol/L) improved COD and NH4 + removal efficiencies by up to 90%. Moreover, the addition of AHLs augmented polysaccharide secretion but had little effect on protein secretion. In comparison to the AS system, the AB system demonstrated promise in terms of pollution removal efficiency and microbial diversity enhancement. PRACTITIONER POINTS: Oscillatoria can serve as an excellent biological carrier for immobilizing bacteria. AHLs (10-9mol/L) regulated the operation state of algae-bacteria symbiotic system. AHLs altered the composition and content of EPS in the algae-bacteria system.

Plant hormone functions and interactions in biological systems

Plant hormone functions and interactions in biological systems

On the outside looking in: roles of endogenous and exogenous strigolactones.

A collection of small molecules called strigolactones (SLs) act as both endogenous hormones to control plant development and as ecological communication cues between organisms. SL signalling overlaps with that of a class of smoke-derived compounds, karrikins (KARs), which have distinct yet overlapping developmental effects on plants. Although the roles of SLs in shoot and root development, in the promotion of arbuscular mycorrhizal (AM) fungal branching and in parasitic plant germination have been well characterized, recent data have illustrated broader roles for these compounds in the rhizosphere. Here, we review the known roles of SLs in development, growth of AM fungi and germination of parasitic plants to develop a framework for understanding the use of SLs as molecules of communication in the rhizosphere. It appears, for example, that there are many connections between SLs and phosphate utilization. Low phosphate levels regulate SL metabolism and, in turn, SLs sculpt root and shoot architecture to coordinate growth and optimize phosphate uptake from the environment. Plant-exuded SLs attract fungal symbionts to deliver inorganic phosphate (Pi) to the host. These and other examples suggest the boundary between exogenous and endogenous SL functions can be easily blurred and a more holistic view of these small molecules is likely to be required to fully understand SL biology. Related to this, we summarize and discuss evidence for a primitive role of SLs in moss as a quorum sensing-like molecule, providing a unifying concept of SLs as endogenous and exogenous signalling molecules.

Receptor-Like Protein Kinases Function Upstream of MAPKs in Regulating Plant Development.

Mitogen-activated protein kinases (MAPKs) are a group of protein kinase broadly involved in various signal pathways in eukaryotes. In plants, MAPK cascades regulate growth, development, stress responses and immunity by perceiving signals from the upstream regulators and transmitting the phosphorylation signals to the downstream signaling components. To reveal the interactions between MAPK cascades and their upstream regulators is important for understanding the functional mechanisms of MAPKs in the life span of higher plants. Typical receptor-like protein kinases (RLKs) are plasma membrane-located to perceive endogenous or exogenous signal molecules in regulating plant growth, development and immunity. MAPK cascades bridge the extracellular signals and intracellular transcription factors in many RLK-mediated signaling pathways. This review focuses on the current findings that RLKs regulate plant development through MAPK cascades and discusses questions that are worth investigating in the near future.

Enhancement of adaptive response in peanut hairy root by exogenous signalling molecules under cadmium stress

Plants counteract Cd toxicity by activating cellular stress responses. The simultaneous exogenous application of methyl jasmonate (MeJA) and methyl-β-cyclodextrin (CD) before Cd exposure improved the response of Arachis hypogaea hairy root culture to the unfavourable effects of Cd toxicity. At 24 h after elicitation, genes that encode key enzymes in the phenylpropanoid biosynthesis pathway (i.e., PAL and RS3) were up-regulated to 3.2- and 5.4-fold changes respectively, thereby inducing stilbene production. The up-regulation of genes that encode transcription factors (i.e., ERF1 and ERF6) significantly increased the expression of several genes (PR4A, PR5, PR10, and chitinase) that encode the pathogenesis-related (PR) proteins to 25.8-, 45-, 5- and 12.6-fold changes, respectively. The more dramatic up-regulation of PR protein-encoding genes demonstrated the significant role of defence proteins in plant protective mechanisms. The prolonged (i.e., 72-h) treatment with MeJA + CD_Cd triggered adaptive responses by substantially increasing the levels of antioxidants, stilbenes, and other phenolic substances. These findings suggest that the interaction between signalling elicitors (MeJA and CD) and Cd modulates a complex signalling network for plant defence system.

Exogenous Signaling Molecules Released from Aptamer-Functionalized Hydrogels Promote the Survival of Mesenchymal Stem Cell Spheroids.

Mesenchymal stem cells (MSCs) have a very low survival rate after in vivo delivery, which limits their great promise for treating human diseases. Various strategies have been studied to overcome this challenge. However, an overlooked but important potential is to apply exogenous signaling molecules as biochemical cues to promote MSC survival, presumably because it is well-known that MSCs themselves can release a variety of potent signaling molecules. Thus, the purpose of this work was to examine and understand whether the release of exogenous signaling molecules from hydrogels can promote the survival of MSC spheroids. Our data show that more vascular endothelial growth factor (VEGF) but not platelet-derived growth factor BB (PDGF-BB) were released from MSC spheroids in comparison with 2D cultured MSCs. Aptamer-functionalized fibrin hydrogel (aFn) could release exogenous VEGF and PDGF-BB in a sustained manner. PDGF-BB-loaded aFn promoted MSC survival by ∼70% more than VEGF-loaded aFn under the hypoxic condition in vitro. Importantly, PDGF-BB-loaded aFn could double the survival rate of MSC spheroids in comparison with VEGF-loaded aFn during the one-week test in vivo. Therefore, this work demonstrated that defined exogenous signaling molecules (e.g., PDGF-BB) can function as biochemical cues for promoting the survival of MSC spheroids in vivo.

Bioinspired Precision Engineering of Three-Dimensional Epithelial Stem Cell Microniches.

Maintenance of the epithelium relies on stem cells residing within specialized microenvironments, known as epithelial crypts. Two-photon polymerization (2PP) is a valuable tool for fabricating 3D micro/nanostructures for stem cell niche engineering applications. Herein, biomimetic gelatin methacrylate-based constructs, replicating the precise geometry of the limbal epithelial crypt structures (limbal stem cell "microniches") as an exemplar epithelial niche, are fabricated using 2PP. Human limbal epithelial stem cells (hLESCs) are seeded within the microniches in xeno-free conditions to investigate their ability to repopulate the crypts and the expression of various differentiation markers. Cell proliferation and a zonation in cell phenotype along the z-axis are observed without the use of exogenous signaling molecules. Significant differences in cell phenotype between cells located at the base of the microniche and those situated towards the rim are observed, demonstrating that stem cell fate is strongly influenced by its location within a niche and the geometrical details of where it resides. This study provides insight into the influence of the niche's spatial geometry on hLESCs and demonstrates a flexible approach for the fabrication of biomimetic crypt-like structures in epithelial tissues. This has significant implications for regenerative medicine applications and can ultimately lead to implantable synthetic "niche-based" treatments.

A CBL-interacting protein kinase AdCIPK5 confers salt and osmotic stress tolerance in transgenic tobacco

CBL interacting protein kinases play important roles in adaptation to stress conditions. In the present study, we isolated a CBL-interacting protein kinase homolog (AdCIPK5) from a wild peanut (Arachis diogoi) with similarity to AtCIPK5 of Arabidopsis. Expression analyses in leaves of the wild peanut showed AdCIPK5 induction by exogenous signaling molecules including salicylic acid, abscisic acid and ethylene or abiotic stress factors like salt, PEG and sorbitol. The recombinant AdCIPK5-GFP protein was found to be localized to the nucleus, plasma membrane and cytoplasm. We overexpressed AdCIPK5 in tobacco plants and checked their level of tolerance to biotic and abiotic stresses. While wild type and transgenic plants displayed no significant differences to the treatment with the phytopathogen, Phytophthora parasitica pv nicotianae, the expression of AdCIPK5 increased salt and osmotic tolerance in transgenic plants. Analysis of different physiological parameters revealed that the transgenic plants maintained higher chlorophyll content and catalase activity with lower levels of H2O2 and MDA content during the abiotic stress conditions. AdCIPK5 overexpression also contributed to the maintenance of a higher the K+/Na+ ratio under salt stress. The enhanced tolerance of transgenic plants was associated with elevated expression of stress-related marker genes; NtERD10C, NtERD10D, NtNCED1, NtSus1, NtCAT and NtSOS1. Taken together, these results indicate that AdCIPK5 is a positive regulator of salt and osmotic stress tolerance.

Activation of paulomycin production by exogenous γ-butyrolactone signaling molecules in Streptomyces albidoflavus J1074.

The interspecies communication roles of γ-butyrolactones (GBLs) have been described for a long time but are still poorly understood. Herein, we analyzed more than 1000 Streptomyces strains and noticed a big quantitative gap between the strains with GBL biosynthetic genes and the strains with GBL receptor genes, which implies the wide-spread of GBLs as interspecies signals in Streptomyces and their great potential in the activation of silent natural product gene clusters. Streptomyces albidoflavus J1074, which has one GBL receptor gene but no GBL biosynthetic gene, was chosen as a target to study the possible interspecies communication roles of GBLs. At first, the GBL biosynthetic genes from Streptomyces coelicolor M145 were expressed in S. albidoflavus J1074, which enabled the S. albidoflavus strains to synthesize Streptomyces coelicolor butanolides (SCBs) and activated the production of paulomycins. Further studies showed that this activation process requires the participation of the GBL receptor gene XNR_4681. The results suggest that the expression of exogenous GBL biosynthetic genes can modulate the metabolisms of GBL non-producing strains, and this regulation role might be meaningful for silent gene cluster activation in Streptomyces. At final, we synthesized racemic-SCB2 and tried to simplify the activation process by adding SCB2 directly to S. albidoflavus J1074, which unfortunately failed to induce paulomycin production.

Role of exogenous signaling molecules in alleviating salt-induced oxidative stress in rice (Oryza sativa L.): a comparative study

Intensified salt stress is an acute hindrance to crop cultivation, whereas plant signaling molecules can efficiently prompt salinity tolerance. Therefore, this study was accomplished to explore the potential salinity stress-mitigating effect of different signaling molecules in rice. The rice (cv. BRRI dhan29) seeds were immersed in 20 mM KNO3, 0.15 mM H2O2, 0.8 mM AsA (ascorbic acid) and 10 mM CaCl2 solutions for 24 h. Eventually, primed seeds were exposed to 75 mM NaCl in Petri dishes during germination. Moreover, 14-day-old rice seedlings were pretreated with different agents, viz., KNO3, H2O2, AsA and CaCl2 (concentrations were same as previous), for 2 days. Primed and non-primed seedlings were grown for 4 days under 75 mM NaCl stress condition. The result revealed that salt stress caused reduced germination indices and pre-seedling and seedling growth inhibition and impaired photosynthetic capacity, whereas catalase (CAT), ascorbate peroxidase (APX) and peroxidase (POX) activities were decreased in salt-treated plants. However, application of the four signaling molecules promoted the germination indices and growth and resisted chlorosis. Pretreatment with CaCl2 and AsA was observed to be relatively more efficient in conferring salinity tolerance of rice as reflected from the significant enhanced germination and growth in the saline medium by increasing reactive oxygen species (ROS) scavenging capacity, both at germination and seedling stage. All the selected signaling molecules significantly detoxified excess ROS, i.e., H2O2 and $${\text{O}}_{2}^{ \cdot - }$$ and reduced lipid peroxidation by up-regulating the enzymes, CAT, APX and POX. Moreover, H2O2 and KNO3 pretreatment also mitigated salt-imposed oxidative stress and enhanced growth performance of rice seedlings. Overall, the study confirms that CaCl2 and AsA pretreatment were more effective than H2O2 and KNO3 priming to improve salt tolerance in rice.

Elicitation of Isatis tinctoria L. hairy root cultures by salicylic acid and methyl jasmonate for the enhanced production of pharmacologically active alkaloids and flavonoids

Plant cell and organ cultures via the implementation of effective elicitation strategies can offer attractive biotechnological platforms for the enhanced production of phytochemicals of pharmaceutical interest. For the first time, the elicitation of exogenous signal molecules was conducted to enhance the production of pharmacologically active alkaloids and flavonoids in Isatis tinctoria L. hairy root cultures (ITHRCs). ITHRCs III and V correspondingly possessing high alkaloid and flavonoid productivity were adopted for elicitation treatments. The maximum accumulation of alkaloids in ITHRCs III elicited by 142.61 µM salicylic acid for 28.18 h and flavonoids in ITHRCs V elicited by 179.54 µM methyl jasmonate for 41.87 h increased 5.89- and 11.21-folds as compared with controls, respectively. Moreover, expressions of 11 genes involved in alkaloid and flavonoid biosynthetic pathways were significantly up-regulated following elicitation, among which YUCCA, CHI and F3′H genes might play a crucial role in the target phytochemical augmentation. Overall, two effective elicitation protocols were provided here to improve the yields of bioactive alkaloids and flavonoids in ITHRCs, which was useful for the scale-up production of these valuable compounds to meet the demands for natural bioactive ingredients by pharmaceutical industries.