Endophytic Bacillus pumilus G5 Interacting with Silicon to Improve Drought Stress Resilience in Glycyrrhiza uralensis Fisch. by Modulating Nitrogen Absorption, Assimilation, and Metabolism Pathways.

Drought stress has become the primary severe threat to global agriculture production, including medicinal plants. Plant growth-promoting bacteria (PGPB) and environmentally friendly element silicon (Si) have emerged as effective methods in alleviating drought stress in various plants. Here, the effects of the plant endophytic G5 interaction with Si on regulating nitrogen absorption, assimilation, and metabolism pathways were investigated in the morphophysiological and gene attributes of Glycyrrhiza uralensis exposed to drought. Results showed that G5+Si application improved nitrogen absorption and assimilation by increasing the available nitrogen content in the soil, further improving the nitrogen utilization efficiency. Then, G5+Si triggered the accumulation of the major adjustment substances proline, γ-aminobutyric acid, putrescine, and chlorophyll, which played an important role in contributing to maintaining balance and energy supply in G. uralensis exposed to drought. These findings will provide new ideas for the combined application of PGPR and Si on both soil and plant systems in a drought habitat.

Bacillus cereus G2 alleviate salt stress in Glycyrrhiza uralensis Fisch. by balancing the downstream branches of phenylpropanoids and activating flavonoid biosynthesis.

The salinity environment is one of the biggest threats to Glycyrrhiza uralensis Fisch. (G. uralensis) growth, resulting from the oxidative stress caused by excess reactive oxygen species (ROS). Flavonoids are the main pharmacodynamic composition and help maintain ROS homeostasis and mitigate oxidative damage in G. uralensis in the salinity environment. To investigate whether endophytic Bacillus cereus G2 can improve the salt-tolerance of G. uralensis through controlling flavonoid biosynthesis, the transcriptomic and physiological analysis of G. uralensis treated by G2 in the saline environment was conducted, focused on flavonoid biosynthesis-related pathways. Results uncovered that salinity inhibited flavonoids synthesis by decreasing the activities of phenylalanine ammonialyase (PAL) and 4-coumarate-CoA ligase (4CL) (42% and 39%, respectively) due to down-regulated gene Glyur000910s00020578 at substrate level, and then decreasing the activities of chalcone isomerase (CHI) and chalcone synthase (CHS) activities (50% and 42%, respectively) due to down-regulated genes Glyur006062s00044203 and Glyur000051s00003431, further decreasing isoliquiritigenin content by 53%. However, salt stress increased liquiritin content by 43%, which might be a protective mechanism of salt-treated G. uralensis seedlings. Interestingly, G2 enhanced PAL activity by 27% whereas reduced trans-cinnamate 4-monooxygenase (C4H) activity by 43% which could inhibit lignin biosynthesis but promote flavonoid biosynthesis of salt-treated G. uralensis at the substrate level. G2 decreased shikimate O-hydroxycinnamoyltransferase (HCT) activity by 35%, increased CHS activity by 54% through up-regulating the gene Glyur000051s00003431 encoding CHS, and increased CHI activity by 72%, thereby decreasing lignin (34%) and liquiritin (24%) content, but increasing isoliquiritigenin content (35%), which could mitigate oxidative damage and changed salt-tolerance mechanism of G. uralensis.

Integrated transcriptomics and metabolomics reveal specific phenolic and flavonoid accumulation in licorice (Glycyrrhiza uralensis Fisch.) induced by arbuscular mycorrhiza symbiosis under drought stress.

Arbuscular mycorrhizal (AM) symbiosis can strengthen plant defense against abiotic stress, such as drought, through multiple mechanisms; however, the specialized chemical defenses induced by AM symbiosis are largely unknown. In a pot experiment, licorice (Glycyrrhiza uralensis Fisch.) inoculated with and without arbuscular mycorrhizal fungus Rhizophagus irregularis Schenck & Smith were grown under well-watered or water deficit conditions. Transcriptomic and metabolomic analyses were combined to investigate licorice root specialized metabolism induced by AM symbiosis under drought stress. Results showed that mycorrhizal plants had few dead leaves, less biomass reduction, and less differentially expressed genes and metabolite features in response to drought compared with nonmycorrhizal plants. Transcriptomic and metabolomic data revealed that mycorrhizal roots generally accumulated lignin regardless of the water regime; however, the expression of genes involved in lignin biosynthesis was significantly downregulated by drought stress in mycorrhizal plants. By contrast, AM inoculation significantly decreased specialized metabolites accumulation, including phenolics and flavonoids under well-watered conditions, whereas these decreases turned to be nonsignificant under drought stress. Moreover, these specific phenolics and flavonoids showed significant drought-induced accumulation pattern in mycorrhizal roots. These results highlight that accumulation of specific root phenolics and flavonoids may support the drought tolerance of mycorrhizal plants.

Bacillus cereus G2 Facilitates N Cycle in Soil, Further Improves N Uptake and Assimilation, and Accelerates Proline and Glycine Betaine Metabolisms of Glycyrrhiza uralensis Subjected to Salt Stress.

Soil salinity is a severe abiotic stress that reduces crop productivity. Recently, there has been growing interest in the application of microbes, mainly plant-growth-promoting bacteria (PGPB), as inoculants for saline land restoration and plant salinity tolerance. Herein, the effects of the plant endophyte G2 on regulating soil N cycle, plant N uptake and assimilate pathways, proline and glycine betaine biosynthesis, and catabolic pathways were investigated in Glycyrrhiza uralensis exposed to salinity. The results indicated that G2 improved the efficiency of N absorption and assimilation of plants by facilitating soil N cycling. Then, G2 promoted the synthesis substrates of proline and glycine betaine and accelerated its synthesis rate, which increased the relative water content and reduced the electrolyte leakage, eventually protecting the membrane system caused by salt stress in G. uralensis. These findings will provide a new idea from soil to plant systems in a salinity environment.

Neuroprotective effect of total flavonoids in stems and leaves of Glycyrrhiza uralensis Fisch. on oxidative stress in HT-22 cells and Caenorhabditis elegans.

The Glycyrrhiza uralensis Fisch. is a common traditional Chinese medicine. However, its aerial part is currently not widely studied and used. Therefore, we aimed to investigate the neuroprotective effects of total flavonoids in aerial stems and leaves of Glycyrrhiza uralensis Fisch. (GSF) by an in vitro LPS-induced HT-22 cell model and an in vivo Caenorhabditis elegans (C. elegans) model. In this study, cell apoptosis was evaluated by CCK-8 and Hoechst 33258 staining in LPS-induced HT-22 cells. Meanwhile, ROS level, mitochondrial membrane potential (MMP), and Ca2+ level were detected by the flow cytometer. In vivo, C. elegans was also investigated the effect of GSF on lifespan, spawning, and paralysis. Moreover, the survival of C. elegans to oxidative stimuli (juglone and H2O2), and the nuclear translocation of DAF-16 and SKN-1 were evaluated. The results showed that GSF could inhibit LPS-induced HT-22 cell apoptosis. Moreover, GSF decreased the levels of ROS, MMP, Ca2+, and malondialdehyde (MDA) and increased the activities of SOD and CAT in HT-22 cells. Furthermore, GSF did not affect the lifespan and laying of eggs of C. elegans N2. However, it delayed paralysis in C. elegans CL4176 in a dose-dependent manner. Meanwhile, GSF increased the survival rate of C. elegans CL2006 after juglone and H2O2 treatment, increased SOD and CAT, and decreased MDA levels. Importantly, GSF promoted the nuclear translocation of DAF-16 and SKN-1 in C. elegans TG356 and LC333, respectively. Taken together, GSF can play a protective role in neuronal cells by inhibiting oxidative stress.

Inhibition of Soluble Epoxide Hydrolase Activity by Components of Glycyrrhiza uralensis.

Soluble epoxide hydrolase (sEH) is a target enzyme for the treatment of inflammation and cardiovascular disease. A Glycyrrhiza uralensis extract exhibited ~50% inhibition of sEH at 100 µg/mL, and column chromatography yielded compounds 1-11. Inhibitors 1, 4-6, 9, and 11 were non-competitive; inhibitors 3, 7, 8, and 10 were competitive. The IC50 value of inhibitor 10 was below 2 µM. Molecular simulation was used to identify the sEH binding site. Glycycoumarin (10) requires further evaluation in cells and animals.

Abiotic factors and endophytes co-regulate flavone and terpenoid glycoside metabolism in Glycyrrhiza uralensis.

Recently, endorhizospheric microbiota is realized to be able to promote the secondary metabolism in medicinal plants, but the detailed metabolic regulation metabolisms and whether the promotion is influenced by environmental factors are unclear yet. Here, the major flavonoids and endophytic bacterial communities in various Glycyrrhiza uralensis Fisch. roots collected from seven distinct places in northwest China, as well as the edaphic conditions, were characterized and analyzed. It was found that the soil moisture and temperature might modulate the secondary metabolism in G. uralensis roots partially through some endophytes. One rationally isolated endophyte Rhizobium rhizolycopersici GUH21 was proved to promote the accumulation of isoliquiritin and glycyrrhizic acid significantly in roots of the potted G. uralensis under the relatively high-level watering and low temperature. Furthermore, we did the comparative transcriptome analysis of G. uralensis seedling roots in different treatments to investigate the detailed mechanisms of the environment-endophyte-plant interactions and found that the low temperature went hand in hand with the high-level watering to activate the aglycone biosynthesis in G. uralensis, while GUH21 and the high-level watering cooperatively promoted the in planta glucosyl unit production. Our study is of significance for the development of methods to rationally promote the medicinal plant quality. KEY POINTS: • Soil temperature and moisture related to isoliquiritin contents in Glycyrrhiza uralensis Fisch. • Soil temperature and moisture related to the hosts' endophytic bacterial community structures. • The causal relation among abiotic factors-endophytes-host was proved through the pot experiment.

Multi-Omics Analysis Provides Crucial Insights into the Drought Adaptation of Glycyrrhiza uralensis Fisch.

Drought adaptation of plants is closely related to resistance and tolerance to drought stress as well as the ability to recover after the elimination of the stress. Glycyrrhiza uralensis Fisch is a commonly applied herb whose growth and development are greatly affected by drought. Here, we provide the first comprehensive analysis of the transcriptomic, epigenetic, and metabolic responses of G. uralensis to drought stress and rewatering. The hyper-/hypomethylation of genes may lead to up-/downregulated gene expression, and epigenetic changes can be regarded as an important regulatory mechanism of G. uralensis under drought stress and rewatering. Moreover, integrated transcriptome and metabolome analysis revealed that genes and metabolites involved in pathways of antioxidation, osmoregulation, phenylpropanoid biosynthesis, and flavonoid biosynthesis may regulate the drought adaptation of G. uralensis. This work provides crucial insights into the drought adaptation of G. uralensis and offers epigenetic resources for cultivating G. uralensis with high drought adaptation.

Chromosome-scale genome assembly of Glycyrrhiza uralensis revealed metabolic gene cluster centred specialized metabolites biosynthesis.

A high-quality genome assembly is imperative to explore the evolutionary basis of characteristic attributes that define chemotype and provide essential resources for a molecular breeding strategy for enhanced production of medicinal metabolites. Here, using single-molecule high-fidelity (HiFi) sequencing reads, we report chromosome-scale genome assembly for Chinese licorice (Glycyrrhiza uralensis), a widely used herbal and natural medicine. The entire genome assembly was achieved in eight chromosomes, with contig and scaffold N50 as 36.02 and 60.2 Mb, respectively. With only 17 assembly gaps and half of the chromosomes having no or one assembly gap, the presented genome assembly is among the best plant genomes to date. Our results showed an advantage of using highly accurate long-read HiFi sequencing data for assembling a highly heterozygous genome including its complexed repeat content. Additionally, our analysis revealed that G. uralensis experienced a recent whole-genome duplication at approximately 59.02 million years ago post a gamma (γ) whole-genome triplication event, which contributed to its present chemotype features. The metabolic gene cluster analysis identified 355 gene clusters, which included the entire biosynthesis pathway of glycyrrhizin. The genome assembly and its annotations provide an essential resource for licorice improvement through molecular breeding and the discovery of valuable genes for engineering bioactive components and understanding the evolution of specialized metabolites biosynthesis.

The comparison between the effect of Glycyrrhizae uralensis (Gan-Cao) and Montelukast on the expression of T-bet and GATA-3 genes in children with allergic asthma.

Pediatric allergic asthma is a chronic disease that affects the lungs and airways. If a child is exposed to certain stimulants such as pollen inhalation, colds, or respiratory infections, the lungs become inflamed and if left untreated can lead to dangerous asthma attacks. One of the most important treatments for this disease is the use of leukotriene modulators, such as montelukast. But recently, due to easier access, cheaper prices and fewer side effects, attention has shifted to non-chemical treatments. Gan-Cao (Glycyrrhizae uralensis), as traditional Chinese medicine, has been proved to have a good therapeutic effect on experimental allergic asthma. But its anti-asthma mechanism is currently unclear. Therefore, the study aimed the comparison between the effect of Gan-Cao and montelukast on the expression of T-bet and GATA-3 genes in children with allergic asthma. For this purpose, fifty children with allergic asthma were divided into two groups. The first group was treated with montelukast for one month. The second group was treated with Gan-Cao root extract. Then the peripheral blood mononuclear cells were isolated, their RNA was extracted, and the relative expression of T-bet and GATA3 transcription factors was evaluated by Real-time PCR. The relationship between them and risk factors for asthma was assessed by relevant statistical tests. The result showed the expression of the GATA3 gene (P = 0.102), T-bet gene (P = 0.888), and the expression ratio of T-bet/GATA-3 genes (P = 0.061) was not significantly different between the two groups. It showed that Gan-Cao can affect the expression of these genes just as much as montelukast. Therefore, this Chinese herb can be used as an alternative or supplement medicine to treat allergic asthma in children.

Multi-omics profiling reveals comprehensive microbe-plant-metabolite regulation patterns for medicinal plant Glycyrrhiza uralensis Fisch.

Glycyrrhiza uralensis Fisch is a medicinal plant widely used to treat multiple diseases in Europe and Asia, and its efficacy largely depends on liquiritin and glycyrrhizic acid. The regulatory pattern responsible for the difference in efficacy between wild and cultivated G. uralensis remains largely undetermined. Here, we collected roots and rhizosphere soils from wild (WT) G. uralensis as well as those farmed for 1 year (C1) and 3 years (C3), generated metabolite and transcript data for roots, microbiota data for rhizospheres and conducted comprehensive multi-omics analyses. We updated gene structures for all 40 091 genes in G. uralensis, and based on 52 differentially expressed genes, we charted the route-map of both liquiritin and glycyrrhizic acid biosynthesis, with genes BAS, CYP72A154 and CYP88D6 critical for glycyrrhizic acid biosynthesis being significantly expressed higher in wild G. uralensis than in cultivated G. uralensis. Additionally, multi-omics network analysis identified that Lysobacter was strongly associated with CYP72A154, which was required for glycyrrhizic acid biosynthesis. Finally, we developed a holistic multi-omics regulation model that confirmed the importance of rhizosphere microbial community structure in liquiritin accumulation. This study thoroughly decoded the key regulatory mechanisms of liquiritin and glycyrrhizic acid, and provided new insights into the interactions of the plant's key metabolites with its transcriptome, rhizosphere microbes and environment, which would guide future cultivation of G. uralensis.

Glycyrrhiza uralensis promote the metabolism of toxic components of Aconitum carmichaeli by CYP3A and alleviate the development of chronic heart failure.

Aconitum, as "the first drug of choice for invigorating Yang and saving lives", has been widely used for the treatment of heart failure. However, toxic components of Aconitum can easily lead to serious arrhythmia, even death (Y. CT., 2009; Zhang XM., 2018). In this study, a High Performance Liquid Chromatography (HPLC) method for the determination of aconitine (AC), mesaconitine (MA) and hypaconitine (HA) was established; The effect of Glycyrrhiza on CYP3A1 / 2 mRNA expression was detected by RT-PCR; SD rats were given Aconitum and compatibility of Glycyrrhizae and Aconitum by gavage respectively, the blood concentration of toxic components were determined by LC-MS / MS; The CHF rat model was established by intraperitoneal injection of adriamycin (2.5 mg / kg), and were randomly divided into model, Aconitum, the compatibility of Glycyrrhizae and Aconitum and Captopril group, 5 mice/group. After 4 weeks of gavage, the corresponding indexes were detected by ELISA and HPLC. The results showed that Ketoconazole significantly inhibited the metabolites of AC, MA and HA; Glycyrrhiza induced CYP3A gene expression; The level of ALD in the compatibility of Glycyrrhizae and Aconitum group was significantly lower than that in Aconitum group. After intervention with the compatibility of Glycyrrhizae and Aconitum, ATP increased, ADP decreased significantly. In conclusion, we found Glycyrrhiza promoted the metabolism of toxic components of Aconitum by up regulating the expression of CYP3A, and reduced the content of BNP, Ang II and ALD, improved the energy metabolism disorder of myocardium, alleviated the development of CHF.

Polysaccharides derived from Astragalus membranaceus and Glycyrrhiza uralensis improve growth performance of broilers by enhancing intestinal health and modulating gut microbiota.

This study was conducted to investigate the effects of dietary supplementation of polysaccharides derived from Astragalus membranaceus and Glycyrrhiza uralensis on growth performance, intestinal health, and gut microbiota composition in broilers. A total of 480 one-day-old male Arbor Acres broilers were randomly divided into 4 treatments with 6 replicates comprising 20 broilers each. Treatments included: basal diet without antibiotics (CON); basal diet supplemented with 500 mg/kg terramycin calcium (ANT); basal diet supplemented with 300 mg/kg Astragalus membranaceus polysaccharides (APS); and basal diet supplemented with 150 mg/kg Glycyrrhiza uralensis polysaccharides (GPS). The results showed that ANT, AP,S and GPS supplementation significantly increased average daily gain (ADG) and decreased feed conversion ratio (FCR) of broilers from 1 to 42 d of age. At 42 d, serum immunoglobulin A (IgA), immunoglobulin M (IgM) and immunoglobulin G (IgG) levels of the APS and GPS group were notably higher than those of the CON group, while serum levels of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6) as well as diamine oxidase (DAO) activity in the APS and GPS group were obviously decreased. Moreover, diets supplemented with APS and GPS could significantly increase villus height (VH) and the ratio of villus height to crypt depth (VH/CD) and remarkably upregulated occludin, claudin-1 and mucin-2 (MUC2) mRNA expression in duodenum, jejunum, and ileum of broilers. In addition, 16S rRNA gene sequencing revealed that APS and GPS supplementation altered cecal microbial diversity and composition in broilers. Higher Shannon index was observed in the APS and GPS group compared with the CON group, while GPS supplementation could also increase Chao1 index and Observed species. The result of Principal coordinate analysis (PCoA) showed that microbial community in the CON, ANT, APS, and GPS group clustered separately. Notably, both APS and GPS supplementation significantly decreased the abundance of Bacteroidetes, Bacteroides, Faecalibacterium, Desulfovibrio, and Butyricicoccus, while increased the abundance of Firmicutes, Prevotella, Parabacteroides, Ruminococcus, and Alistipes. The correlation analysis showed that the changes in cecal microbial composition induced by dietary APS and GPS supplementation were closely associated with the alteration of the phenotype of broilers including ADG, FCR, TNF-α, IL-1ß, IL-6, IgA, IgG, DAO, Occludin, Claudin-1, ZO-1, and MUC2. In conclusion, polysaccharides derived from Astragalus membranaceus and Glycyrrhiza uralensis could improve growth performance of broilers by enhancing intestinal health and modulating gut microbiota.

Integrative analysis of the pharmaceutical active ingredient and transcriptome of the aerial parts of Glycyrrhiza uralensis under salt stress reveals liquiritin accumulation via ABA-mediated signaling.

The aerial parts of Glycyrrhiza uralensis supply substantial raw material for the extraction of active pharmaceutical ingredients comprehensively utilized in many industries. Our previous study indicated that salt stress increased the content of active ingredients. However, the regulatory mechanism remains unclear. In this study, RNA-sequencing (RNA-seq) of the aerial parts of G. uralensis treated with 150 mM NaCl for 0, 2, 6, and 12 h was performed to identify the key genes and metabolic pathways regulating pharmacological active component accumulation. The main active component detection showed that liquiritin was the major ingredient and exhibited more than a ten-fold significant increase in the 6 h NaCl treatment. Temporal expression analysis of the obtained 4245 differentially expressed genes (DEGs) obtained by RNA-seq revealed two screened profiles that included the significant up-regulated DEGs (UDEGs) at different treatment points. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of these UDEGs identified phenylpropanoid metabolism and flavonoid biosynthesis as the most significantly enriched pathways in 2 h treated materials. Interestingly, the carotenoid biosynthesis pathway that is related to ABA synthesis was also discovered, and the ABA content was significantly promoted after 6 h NaCl treatment. Following ABA stimulation, the content of liquiritin demonstrated a significant and immediate increase after 2 h treatment, with the corresponding consistent expression of genes involved in the pathways of ABA signal transduction and flavonoid biosynthesis, but not in the pathway of glycyrrhizic acid biosynthesis. Our study concludes that salt stress might promote liquiritin accumulation through the ABA-mediated signaling pathway, and provides effective reference for genetic improvement and comprehensive utilization of G. uralensis.

Diversity and function of rhizosphere microorganisms between wild and cultivated medicinal plant Glycyrrhiza uralensis Fisch under different soil conditions.

Glycyrrhiza uralensis Fisch is a widely cultivated traditional Chinese medicine plant. In the present study, culture-independent microbial diversity analysis and functional prediction of rhizosphere microbes associated with wild and cultivated G. uralensis Fisch plant (collected from two locations) were carried. Soil physicochemical parameters were tested to assess their impact on microbial communities. A total of 4428 OTUs belonging to 41 bacterial phyla were identified. In general, cultivated sample sites were dominated by Actinobacteria whereas wild sample sites were dominated by Proteobacteria. The alpha diversity analysis showed the observed species number was higher in cultivated soil samples when compared with wild soil samples. In beta diversity analysis, it was noticed that the weighted-unifrac distance of two cultivated samples was closer although the samples were collected from different regions. Functional annotation based on PICRUST and FAPROTAX showed that the nitrogen metabolism pathway such as nitrate reduction, nitrogen fixation, nitrite ammonification, and nitrite respiration were more abundant in rhizosphere microorganisms of wild G. uralensis Fisch. These results also correlate in redundancy analysis results which show correlation between NO3--N and wild samples, which indicated that nitrogen nutrition conditions might be related to the quality of G. uralensis Fisch.

ARPI, ß-AS, and UGE regulate glycyrrhizin biosynthesis in Glycyrrhiza uralensis hairy roots.

KEY MESSAGE: ARPI, ß-AS, and UGE were cloned from G. uralensis and their regulatory effects on glycyrrhizin biosynthesis were investigated. ß-AS and UGE but not ARPI positively regulate the biosynthesis of glycyrrhizin. Glycyrrhiza uralensis Fisch. has been used to treat respiratory, gastric, and liver diseases since ancient China. The most important and widely studied active component in G. uralensis is glycyrrhizin (GC). Our pervious RNA-Seq study shows that GC biosynthesis is regulated by multiple biosynthetic pathways. In this study, three target genes, ARPI, ß-AS, and UGE from different pathways were selected and their regulatory effects on GC biosynthesis were investigated using G. uralensis hairy roots. Our data show that hairy roots knocking out ARPI or UGE died soon after induction, indicating that the genes are essential for the growth of G. uralensis hairy roots. Hairy roots with ß-AS knocked out grew healthily. However, they failed to produce GC, suggesting that ß-AS is required for triterpenoid skeleton formation. Conversely, overexpression of UGE or ß-AS significantly increased the GC content, whereas overexpression of ARPI had no obvious effects on GC accumulation in G. uralensis hairy roots. Our findings demonstrate that ß-AS and UGE positively regulate the biosynthesis of GC.

Succession of endophytic fungi and arbuscular mycorrhizal fungi associated with the growth of plant and their correlation with secondary metabolites in the roots of plants.

BACKGROUND: To decipher the root and microbial interaction, secondary metabolite accumulation in roots and the microbial community's succession model during the plant's growth period demands an in-depth investigation. However, till now, no comprehensive study is available on the succession of endophytic fungi and arbuscular mycorrhizal fungi (AMF) with roots of medicinal licorice plants and the effects of endophytic fungi and AMF on the secondary metabolite accumulation in licorice plant's root. RESULTS: In the current study, interaction between root and microbes in 1-3 years old medicinal licorice plant's root and rhizospheric soil was investigated. Secondary metabolites content in licorice root was determined using high-performance liquid chromatography (HPLC). The composition and diversity of endophytic and AMF in the root and soil were deciphered using high-throughput sequencing technology. During the plant's growth period, as compared to AMF, time and species significantly affected the diversity and richness of endophytic fungi, such as Ascomycota, Basidiomycota, Fusarium, Cladosporium, Sarocladium. The growth period also influenced the AMF diversity, evident by the significant increase in the relative abundance of Glomus and the significant decrease in the relative abundance of Diversispora. It indicated a different succession pattern between the endophytic fungal and AMF communities. Meanwhile, distance-based redundancy analysis and Mantel tests revealed root's water content and secondary metabolites (glycyrrhizic acid, liquiritin, and total flavonoids), which conferred endophytic fungi and AMF diversity. Additionally, plant growth significantly altered soil's physicochemical properties, which influenced the distribution of endophytic fungal and AMF communities. CONCLUSIONS: This study indicated a different succession pattern between the endophytic fungal and AMF communities. During the plant's growth period, the contents of three secondary metabolites in roots increased per year, which contributed to the overall differences in composition and distribution of endophytic fungal and AMF communities. The endophytic fungal communities were more sensitive to secondary metabolites than AMF communities. The current study provides novel insights into the interaction between rhizospheric microbes and root exudates.

Allylic Hydroxylation Activity Is a Source of Saponin Chemodiversity in the Genus Glycyrrhiza.

Licorice (Glycyrrhiza) produces glycyrrhizin, a valuable triterpenoid saponin, which exhibits persistent sweetness and broad pharmacological activities. In the genus Glycyrrhiza, three species, Glycyrrhiza uralensis, Glycyrrhiza glabra and Glycyrrhiza inflata, produce glycyrrhizin as their main triterpenoid saponin, which has a ketone group at C-11. Other Glycyrrhiza species produce mainly oleanane-type saponins, which harbor homoannular or heteroannular diene structures that lack the C-11 ketone. Although the glycyrrhizin biosynthetic pathway has been fully elucidated, the pathway involving saponins with diene structures remains unclear. CYP88D6 from G. uralensis is a key enzyme in glycyrrhizin biosynthesis, catalyzing the sequential two-step oxidation of ß-amyrin at position C-11 to produce 11-oxo-ß-amyrin. In this study, we evaluated the functions of CYP88D6 homologs from the glycyrrhizin-producing species G. glabra and G. inflata and from the non-glycyrrhizin-producing species Glycyrrhiza pallidiflora and Glycyrrhiza macedonica, using yeast engineered to supply ß-amyrin as a substrate. Yeast expressing CYP88D6 homologs from glycyrrhizin-producing species produced 11-oxo-ß-amyrin. However, yeast expressing CYP88D6 homologs (such as CYP88D15) from the non-glycyrrhizin-producing Glycyrrhiza species accumulated oleana-9(11),12-dien-3ß-ol and oleana-11,13(18)-dien-3ß-ol; these diene compounds are non-enzymatic or yeast endogenous enzymatic dehydration derivatives of 11α-hydroxy-ß-amyrin, a direct reaction product of CYP88D15. These results suggest that the activities of CYP88D6 homologs, particularly their ability to catalyze the second oxidation, could influence glycyrrhizin productivity and diversify the chemical structures of saponins in Glycyrrhiza plants. A synthetic biological approach to engineer CYP88D15 could enable the production of pharmacologically active saponins with diene structures, such as saikosaponins, whose biosynthetic pathways have yet to be fully characterized.

Root-associated endophytic bacterial community composition and structure of three medicinal licorices and their changes with the growing year.

BACKGROUND: The dried roots and rhizomes of medicinal licorices are widely used worldwide as a traditional medicinal herb, which are mainly attributed to a variety of bioactive compounds that can be extracted from licorice root. Endophytes and plants form a symbiotic relationship, which is an important source of host secondary metabolites. RESULTS: In this study, we used high-throughput sequencing technology and high-performance liquid chromatography to explore the composition and structure of the endophytic bacterial community and the content of bioactive compounds (glycyrrhizic acid, liquiritin and total flavonoids) in different species of medicinal licorices (Glycyrrhiza uralensis, Glycyrrhiza glabra, and Glycyrrhiza inflata) and in different planting years (1-3 years). Our results showed that the contents of the bioactive compounds in the roots of medicinal licorices were not affected by the species, but were significantly affected by the main effect growing year (1-3) (P < 0.05), and with a trend of stable increase in the contents observed with each growing year. In 27 samples, a total of 1,979,531 effective sequences were obtained after quality control, and 2432 effective operational taxonomic units (OTUs) were obtained at 97% identity. The phylum Proteobacteria, Actinobacteria, Bacteroidetes and Firmicutes, and the genera unified-Rhizobiaceae, Pseudomonas, Novosphingobium, and Pantoea were significantly dominant in the 27 samples. Distance-based redundancy analysis (db-RDA) showed that the content of total flavonoids explained the differences in composition and distribution of endophytic bacterial communities in roots of cultivated medicinal liquorices to the greatest extent. Total soil salt was the most important factor that significantly affected the endophytic bacterial community in soil factors, followed by ammonium nitrogen and nitrate nitrogen. Among the leaf nutrition factors, leaf water content had the most significant effect on the endophytic bacterial community, followed by total phosphorus and total potassium. CONCLUSIONS: This study not only provides information on the composition and distribution of endophytic bacteria in the roots of medicinal licorices, but also reveals the influence of abiotic factors on the community of endophytic bacteria and bioactive compounds, which provides a reference for improving the quality of licorice.

Two responses to MeJA induction of R2R3-MYB transcription factors regulate flavonoid accumulation in Glycyrrhiza uralensis Fisch.

Flavonoids are key components of licorice plant that directly affect its medicinal quality. Importantly, the MYB family of transcription factors serves to regulate the synthesis of flavonoids in plants. The MYB transcription factors represent one of the largest families of transcription factors in plants and play important roles in the process of plant growth and development. MYB gene expression is induced by a number of plant hormones, including the lipid-based hormone jasmonate (JA). Methyl jasmonate (MeJA) is an endogenous plant growth regulator that can induce the JA signaling pathway, which functions to regulate the synthesis of secondary metabolites, including flavonoids. In this study, MeJA was added to licorice cell suspensions, and RNA-seq analysis was performed to identify the differentially expressed genes. As a result, the MYB transcription factors GlMYB4 and GlMYB88 were demonstrated to respond significantly to MeJA induction. Subsequently, the GlMYB4 and GlMYB88 protein were shown to localize to the cell nucleus, and it was verified that GlMYB4 and GlMYB88 could positively regulate the synthesis of flavonoids in licorice cells. Overall, this research helps illustrate the molecular regulation of licorice flavonoid biosynthesis induced by MeJA.