[Construction of ATMT system of Fusarium oxysporum and evaluation of its promoting effect on Glycyrrhiza uralensis].

This study aims to evaluate the in vivo function of Fusarium oxysporum in Glycyrrhiza uralensis by salt tolerance,indoleacetic acid(IAA) production capacity, phosphate-dissolving capacity, and iron carrier production capacity. The stable genetic transformation system of the F. oxysporum was established by Agrobacterium tumefaciens-mediated genetic transformation( ATMT)technology, and the stability and staining efficiency of transformants were detected by the cloning of the marker gene green fluorescent protein(GFP) and the efficiency of ß-glucuronidase staining(GUS). Efficient and stable transformants were selected for restaining G. uralensis and evaluating its influence on the growth of the G. uralensis seedlings. The results show that F. oxysporum has good salt tolerance and could still grow on potato glucose agar(PDA) medium containing 7% sodium chloride, but the growth rate slows down with the increase in sodium chloride content in PDA medium. F. oxysporum has the function of producing indoleacetic acid, and the concentration of IAA in its fermentation broth is about 3. 32 mg · m L~(-1). In this study, the genetic transformation system of F. oxysporum is successfully constructed, and the ATMT system is efficient and stable. One transformant with both high staining efficiency and genetic stability is selected, and the restaining rate of the transformant in G. uralensis is 76. 92%, which could significantly improve the main root length of one-month-old G. uralensis seedlings and promote the growth and development of G. uralensis seedlings. The results of this study can lay the foundation for the development of biological bacterial fertilizer and the growth regulation of high-quality G. uralensis.

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 Metabolomics and Transcriptomics Analyses Highlight the Flavonoid Compounds Response to Alkaline Salt Stress in Glycyrrhiza uralensis Leaves.

Glycyrrhiza uralensis is a saline-alkali-tolerant plant whose aerial parts are rich in flavonoids; however, the role of these flavonoids in saline-alkali tolerance remains unclear. Herein, we performed physiological, metabolomics, and transcriptomics analyses in G. uralensis leaves under alkaline salt stress for different durations. Alkaline salt stress stimulated excessive accumulation of reactive oxygen species and consequently destroyed the cell membrane, causing cell death, and G. uralensis initiated osmotic regulation and the antioxidant system to respond to stress. In total, 803 metabolites, including 244 flavonoids, were detected via metabolomics analysis. Differentially altered metabolites and differentially expressed genes were coenriched in flavonoid-related pathways. Genes such as novel.4890, Glyur001511s00039602, and Glyur000775s00025737 were highly expressed, and flavonoid metabolites such as 2'-hydroxygenistein, apigenin, and 3-O-methylquercetin were upregulated. Thus, flavonoids as nonenzymatic antioxidants play an important role in stress tolerance. These findings provide novel insights into the response of G. uralensis to alkaline salt stress.

Multi-Omics Elucidates Difference in Accumulation of Bioactive Constituents in Licorice (Glycyrrhiza uralensis) under Drought Stress.

Licorice is a frequently applied herb with potential edible and medicinal value based on various flavonoids and triterpenes. However, studies on detailed flavonoid and triterpene metabolism and the molecular basis of their biosynthesis in licorice are very limited, especially under drought conditions. In the present study, we carried out transcriptome, proteome, and metabolome experiments. To ultimately combine three omics for analysis, we performed a bioinformatics comparison, integrating transcriptome data and proteome data through a Cloud platform, along with a simplified biosynthesis of primary flavonoids and triterpenoids in the KEGG pathway based on metabolomic results. The biosynthesis pathways of triterpenes and flavonoids are enriched at both gene and protein levels. Key flavonoid-related genes (PAL, 4CL, CHS, CHI, CYP93C, HIDH, HI4OMT, and CYP81E1_7) and representative proteins (HIDH, CYP81E1_7, CYP93C, and VR) were obtained, which all showed high levels after drought treatment. Notably, one R2R3-MYB transcription factor (Glyur000237s00014382.1), a critical regulator of flavonoid biosynthesis, achieved a significant upregulated expression as well. In the biosynthesis of glycyrrhizin, both gene and protein levels of bAS and CYP88D6 have been found with upregulated expression under drought conditions. Most of the differentially expressed genes (DEGs) and proteins (DEPs) showed similar expression patterns and positively related to metabolic profiles of flavonoid and saponin. We believe that suitable drought stress may contribute to the accumulation of bioactive constituents in licorice, and our research provides an insight into the genetic study and quality breeding in this plant.

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.

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.

Expression profiling of the mitogen-activated protein kinase gene family reveals their diverse response pattern in two different salt-tolerant Glycyrrhiza species.

BACKGROUND: Mitogen-activated protein kinases (MPKs) play important role in response to environmental stress as crucial signal receptors or sensors. Our previous study indicated that salt stress acts as a positive factor to stimulate the production of pharmacodynamic metabolites in the medicinal plant Glycyrrhiza uralensis. Currently, little is known about the MPK gene family and their functions in the medicinal plant G. uralensis. OBJECTIVE: Identification, comprehensive bioinformatic analysis, expression profiling, and response pattern under salt stress of the G. uralensis GuMPK gene family. METHODS: Genome-wide investigation and expression profiling of the MPK gene family in G. uralensis, and their phylogenetic relationships, evolutionary characteristics, gene structure, motif distribution, promoter cis-acting element, and expression pattern under salt stress in two different salt-tolerant Glycyrrhiza species were performed. RESULTS: A total of 20 G. uralensis GuMPK genes were identified and categorized into five groups, and had conserved gene structure and motif distribution. Expression profiling of GuMPK genes suggested their potentially diverse functions in plant growth and in response to phytohormones and environmental stress, particularly GuMPK1, 2, 5, and 10 as key components for G. uralensis in response to abiotic stress. Further expression analysis under NaCl treatment in two different salt-tolerant Glycyrrhiza species displayed the MPKs' different response patterns, emphasizing the role of MPK2, 5, 7, and 16 as potentially crucial genes for Glycyrrhiza to respond to salt stress. CONCLUSION: Our results provide a genome-wide identification and expression profiling of MPK gene family in G. uralensis, and establish the foundation for screening key responsive genes and understanding the potential function and regulatory mechanism of GuMPKs in salt responsiveness.

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.

Molecular cloning and functional characterization of UGTs from Glycyrrhiza uralensis flavonoid pathway.

Glycyrrhiza uralensis Fisch., a well-known medicinal plant, contains flavonoids including liquiritigenin and isoliquiritigenin, and their corresponding glycoside liquiritin and isoliquiritin. Although some genes encoding UDP-glycosyltransferases (UGTs) have been functionally characterized in G. uralensis, other UGTs mechanisms of glycosylation remain to be elucidated. Against this background the aim of the present study included cloning and characterization of two full-length cDNA clones of GuUGT isoforms from the UGT multigene family. These included GuUGT2 (NCBI acc. MK341791) and GuUGT3 (NCBI acc. MK341793) with an ORF of 1473 and 1332 bp, respectively. Multiple alignments and phylogenetic analysis revealed GuUGTs protein of Glycine max had a high homology to that of other plants. Meanwhile, quantitative real-time PCR was performed to detect the transcript levels of GuUGTs in different tissues. The results indicated that GuUGTs was more expressed in roots as compared to the leaves, and significantly up-regulated upon NaCl stress. The recombinant protein was heterologous expressed in Escherichia coli and exhibited a high level of UGT activity, catalyzing formation of isoliquiritin and liquiritin from isoliquiritigenin and liquiritigenin. The key residues of GuUGT2 for liquiritigenin glycosylation (Asn223), isoliquiritigenin (Asp272) were predicted by molecular docking and residue scanning based on simulated mutations. These results could serve as an important reference to understand the function of the UGT family. In addition, the identification of GuUGT2 and GuUGT3 provides a foundation for future studies of flavonoid biosynthesis in G. uralensis.

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.

A cellulose synthase-derived enzyme catalyses 3-O-glucuronosylation in saponin biosynthesis.

Triterpenoid saponins are specialised metabolites distributed widely in the plant kingdom that consist of one or more sugar moieties attached to triterpenoid aglycones. Despite the widely accepted view that glycosylation is catalysed by UDP-dependent glycosyltransferase (UGT), the UGT which catalyses the transfer of the conserved glucuronic acid moiety at the C-3 position of glycyrrhizin and various soyasaponins has not been determined. Here, we report that a cellulose synthase superfamily-derived glycosyltransferase (CSyGT) catalyses 3-O-glucuronosylation of triterpenoid aglycones. Gene co-expression analyses of three legume species (Glycyrrhiza uralensis, Glycine max, and Lotus japonicus) reveal the involvement of CSyGTs in saponin biosynthesis, and we characterise CSyGTs in vivo using Saccharomyces cerevisiae. CSyGT mutants of L. japonicus do not accumulate soyasaponin, but the ectopic expression of endoplasmic reticulum membrane-localised CSyGTs in a L. japonicus mutant background successfully complement soyasaponin biosynthesis. Finally, we produced glycyrrhizin de novo in yeast, paving the way for sustainable production of high-value saponins.

Genome-wide identification of abscisic acid (ABA) receptor pyrabactin resistance 1-like protein (PYL) family members and expression analysis of PYL genes in response to different concentrations of ABA stress in Glycyrrhiza uralensis.

As abscisic acid (ABA) receptor, the pyrabactin resistance 1-like (PYR/PYL) protein (named PYL for simplicity) plays an important part to unveil the signal transduction of ABA and its regulatory mechanisms. Glycyrrhiza uralensis, a drought-tolerant medicinal plant, is a good model for the mechanism analysis of ABA response and active compound biosynthesis. However, knowledge about PYL family in G. uralensis remains largely unknown. Here, 10 PYLs were identified in G. uralensis genome. Characterization analysis indicated that PYLs in G. uralensis (GuPYLs) are relatively conserved. Phylogenetic analysis showed that GuPYL1-3 belongs to subfamily I, GuPYL4-6 and GuPYL10 belong to subfamily II and GuPYL7-9 belongs to subfamily III. In addition, transcriptome data presented various expression levels of GuPYLs under different exogenous ABA stresses. The expression pattern of GuPYLs was verified by Quantitative real-time polymerase chain reaction (qRT-PCR). The study proved that GuPYL4, GuPYL5, GuPYL8 and GuPYL9 genes are significantly up-regulated by ABA stress and the response process is dynamic. This study paves the way for elucidating the regulation mechanism of ABA signal to secondary metabolites and improving the cultivation and quality of G. uralensis using agricultural strategies.

Mining of UDP-glucosyltrfansferases in licorice for controllable glycosylation of pentacyclic triterpenoids.

Pentacyclic triterpenoids have wide applications in the pharmaceutical industry. The precise glucosylation at C-3 OH of pentacyclic triterpenoids mediated by uridine 5'-diphospho-glucosyltransferase (UDP-glucosyltransferase [UGT]) is an important way to produce valuable derivatives with various improved functions. However, most reported UGTs suffer from low regiospecificity toward the OH and COOH groups of pentacyclic triterpenoids, which significantly decreases the reaction efficiency. Here, two new UGTs (UGT73C33 and UGT73F24) were discovered in Glycyrrhiza uralensis. UGT73C33 showed high activity but poor regioselectivity toward the C-3 OH and C-30 COOH of pentacyclic triterpenoid, producing three glucosides. UGT73F24 showed rigid regioselectivity toward C-3 OH of typical pentacyclic triterpenoids producing only C-3 O-glucosylated derivatives. In addition, UGT73C33 and UGT73F24 showed a broad substrate scope toward typical flavonoids with various sugar donors. Next, the substrate recognition mechanism of UGT73F24 toward glycyrrhetinic acid (GA) and UDP-glucose was investigated. Two key residues, I23 and L84, were identified to determine activity, and site-directed mutagenesis of UGT73F24-I23G/L84N increased the activity by 4.1-fold. Furthermore, three in vitro GA glycosylation systems with UDP-recycling were constructed, and high yields of GA-3-O-Glc (1.25 mM), GA-30-O-Glc (0.61 mM), and GA-di-Glc (0.26 mM) were obtained. The de novo biosynthesis of GA-3-O-glucose (26.31 mg/L) was also obtained in engineered yeast.

Integrated Analysis of mRNA and microRNA Elucidates the Regulation of Glycyrrhizic Acid Biosynthesis in Glycyrrhiza uralensis Fisch.

Licorice (Glycyrrhiza) is a staple Chinese herbal medicine in which the primary bioactive compound is glycyrrhizic acid (GA), which has important pharmacological functions. To date, the structural genes involved in GA biosynthesis have been identified. However, the regulation of these genes in G. uralensis has not been elucidated. In this study, we performed a comprehensive analysis based on the transcriptome and small RNAome by high-throughput sequencing. In total, we identified 18 structural GA genes and 3924 transporter genes. We identified genes encoding 2374 transporters, 1040 transcription factors (TFs), 262 transcriptional regulators (TRs) and 689 protein kinases (PKs), which were coexpressed with at least one structural gene. We also identified 50,970 alternative splicing (AS) events, in which 17 structural genes exhibited AS. Finally, we also determined that miRNAs potentially targeted 4 structural genes, and 318, 8, and 218 miRNAs potentially regulated 150 TFs, 34 TRs, and 88 PKs, respectively, related to GA. Overall, the results of this study helped to elucidate the gene expression and regulation of GA biosynthesis in G. uralensis, provided a theoretical basis for the synthesis of GA via synthetic biology, and laid a foundation for the cultivation of new varieties of licorice with high GA content.

A Comprehensive Transcriptome-Wide Identification and Screening of WRKY Gene Family Engaged in Abiotic Stress in Glycyrrhiza glabra.

The study reports 147 full-length WRKY genes based on the transcriptome analysis of Glycyrrhiza genus (G. glabra and G. uralensis). Additional motifs in G. glabra included DivIVA (GgWRKY20) and SerS Superfamily (GgWRKY21) at the C-terminal, and Coat family motifs (GgWRKY55) at the N-terminal of the proteins, while Exo70 exo cyst complex subunit of 338 amino acid (GuWRKY9) was present at the N-terminal of G. uralensis only. Plant Zn cluster super-family domain (17 WRKYs) and bZIP domain (2 WRKYs) were common between the two species. Based on the number of WRKY domains, sequence alignment and phylogenesis, the study identified GuWRKY27 comprising of 3 WRKY domains in G. uralensis and a new subgroup-IIf (10 members), having novel zinc finger pattern (C-X4-C-X22-HXH) in G. glabra. Multiple WRKY binding domains (1-11) were identified in the promoter regions of the GgWRKY genes indicating strong interacting network between the WRKY proteins. Tissue-specific expression of 25 GgWRKYs, under normal and treated conditions, revealed 11 of the 18 induction factor triggered response corroborating to response observed in AtWRKYs. The study identified auxin-responsive GgWRKY 55 & GgWRKY38; GA3 responsive GgWRKYs15&59 in roots and GgWRKYs8, 20, 38, 57 &58 in the shoots of the treated plant. GgWRKYs induced under various stresses included GgWRKY33 (cold), GgWRKY4 (senescence), GgWRKYs2, 28 & 33 (salinity) and GgWRKY40 (wounding). Overall, 23 GgWRKYs responded to abiotic stress, and 17 WRKYs were induced by hormonal signals. Of them 13 WRKYs responded to both suggesting inter-connection between hormone signalling and stress response. The present study will help in understanding the transcriptional reprogramming, protein-protein interaction and cross-regulation during stress and other physiological processes in the plant.

Comparative Proteomic Analysis Reveals the Molecular Mechanisms Underlying the Accumulation Difference of Bioactive Constituents in Glycyrrhiza uralensis Fisch under Salt Stress.

Licorice (Glycyrrhiza uralensis Fisch) possesses a substantial share of the global markets for its unique sweet flavor and diverse pharmacological compounds. Cultivated licorice is widely distributed in northwest regions of China, covered with land with a broad range of salinities. A preliminary study indicated that suitable salt stress significantly increased the content of bioactive constituents in licorice. However, the molecular mechanisms underlying the influence of salinity on the accumulation of these constituents remain unclear, which hinders quality breeding of cultivated licorice. In our study, flavonoid-related structural genes were obtained, and most of them, such as phenylalanine ammonia-lyases, cinnamate 4-hydroxylases, 4-coumarate: CoA ligases, chalcone synthases, chalcone-flavanone isomerase, and flavonol synthase, showed high levels after salt treatment. In the biosynthesis of glycyrrhizin, three key enzymes (bAS, CYP88D6, and CYP72A154) were identified as differentially expressed proteins and remarkably upregulated in the salt-stressed group. Combining these results with the contents of 14 bioactive constituents, we also found that the expression patterns of those structural proteins were logically consistent with changes in bioactive constituent profiles. Thus, we believe that suitable salt stress increased the accumulation of bioactive constituents in licorice by upregulating proteins involved in the related biosynthesis pathways. This work provided valuable proteomic information for unraveling the molecular mechanism of flavonoid and glycyrrhizin metabolism and offered fundamental resources for quality breeding in licorice.

Mining of Sucrose Synthases from Glycyrrhiza uralensis and Their Application in the Construction of an Efficient UDP-Recycling System.

Sucrose synthase (SUS) plays an important role in carbohydrate metabolism in plants. The SUS genes in licorice remain unknown. To reveal the sucrose metabolic pathway in licorice, all the 12 putative SUS genes of Glycyrrhiza uralensis were systematically identified by genome mining, and two novel SUSs (GuSUS1 and GuSUS2) were isolated and characterized for the first time. Furthermore, we found that the flexible N-terminus was responsible for the low stability of plant SUSs, and deletion of redundant N-terminus improved the stability of GuSUS1 and GuSUS2. The half-life of both GuSUS1 and GuSUS2 mutants was increased by 2-fold. Finally, the GuSUS1 mutant was coupled with UGT73C11 for the glycosylation of glycyrrhetinic acid (GA) with uridine 5'-diphosphate disodium salt hydrate (UDP) in situ recycling, and GA conversion was increased by 7-fold. Our study not only identified the SUS genes in licorice but also provided a stable SUS mutant for the construction of an efficient UDP-recycling system for GA glycosylation.

Induction of signal molecules and expression of functional genes after Pichia pastoris stimulation in Glycyrrhiza uralensis Fisch adventitious roots.

Glycyrrhiza uralensis Fisch is threatened by over-development and consumption, and therefore, in urgent need of protection. Elicitation is considered to be an effective strategy to enhance the secondary metabolites in plant cell and organ cultures. Secondary metabolite, signal molecules, and gene expression in adventitious roots were studied by HPLC-ESI-MSn , commercially available kits and qRT-PCR method, respectively. In the present study, with the addition of linolenic acid, linoleic acid, and Pichia pastoris, the highest concentration of metabolites was achieved by P. pastoris treatment. The contents of total flavonoids (7.16 mg/g) and polysaccharide (149.76 mg/g) peaked at 100 mg/L of P. pastoris, which increased by 3.09-fold and 3.28-fold compared with the control, respectively. However, the highest concentration of glycyrrhizic acid (0.62 mg/g) and glycyrrhetinic acid (0.29 mg/g) were obtained in 200 mg/L of P. pastoris and which were 3.89-fold and 2.42-fold more than the control group, respectively. ESI-MSn analysis indicated that licoricesaponine B2, licoricesapoine G2, licoricesaponine J2, ononin, uralenin, gancaonin C were only identified in the P. pastoris treatment group. Furthermore, P. pastoris also enhanced accumulation of salicylic acid, jasmonic acid, nitric oxide and activities of antioxidant enzymes involved in the plant defense response. In addition, the transcriptional activity of genes involved in glycyrrhizic acid biosynthesis was significantly increased under the treatment of P. pastoris. The results provided a scientific evidence for the further exploitation of G. uralensis adventitious roots and clinical medication. PRACTICAL APPLICATIONS: This study provided an effective strategy to enhance metabolites by Pichia pastoris treatment in adventitious roots of G. uralensis. The data provide a scientific evidence for the further exploitation of G. uralensis adventitious roots and clinical medication.