All 29 Glycyrrhiza inflata accessions used were pure Xinjiang germplasms previously collected and authenticated by our laboratory. Seeds underwent 50-min 98% sulfuric acid treatment to break dormancy and enhance germination, followed by thorough rinsing to remove residues. Field cultivation occurred at Shihezi University Experimental Station (44°16’N, 86°00’E) under temperate continental climate (≈170-day frost-free period; July mean: 25–26°C; annual precipitation: 125–207 mm). Seeds were planted in May using drip irrigation (2.05-m wide mulch, six rows per bed, three drip lines) with alternating wide-narrow row spacing (18,000 plants/ha; irrigation: 4,200 m³/ha). Roots (20 cm segments from 5 plants/accession) were harvested during late September–early October for yield assessment (fresh/dry weight) and phytochemical analysis.

Saline-alkali field trials were conducted in Minfeng County (82°22’E, 35°20’N) featuring typical temperate desert climate (mean annual temp: stable; precipitation: 30.5 mm; evaporation: 2,756 mm; frost-free period: 194 days; sunshine: 2,842.2 hr). Identical root sampling protocols were followed.

Germination assays used plump, uniform seeds (50 seeds/dish on filter paper; 3 treatments × 3 replicates) with 10 mL test solution replenished periodically using original concentrations to maintain osmolarity (distilled water control). Salt stress experiments proceeded in climate-controlled chambers (200 μmol·m⁻²·s⁻¹ PPFD; 16/8-h light/dark; 50–55% RH; 28°C/25°C day/night). Seeds were sown in vermiculite-filled pots (10×10×10 cm; 6 plants/pot), watered biweekly and fertilized weekly with half-strength MS medium. At 20-cm height, plants received 150 mM NaCl (pre-optimized concentration). Aboveground and root tissues (harvested at root-shoot junction) were sampled at 0, 2, 6, 12, and 24 h post-treatment (three biological replicates). Samples were rinsed, flash-frozen in liquid nitrogen, and stored at −80 °C for subsequent phytochemical quantification and RNA extraction (transcriptome sequencing).

The measurement of this part was carried out by a third-party institution, the Agricultural Science Research Institute of the 7th Division of Xinjiang Production and Construction Corps. They took five samples from the 4,500 mu of land in Area 1 of the Salayuketek Township, Minfen County, Xinjiang Province, at depths of 0–30 cm and 30–60 cm. The pH measurement results of the 0–30 cm soil samples were 8.27, with water-soluble salts at 5.33 g/kg, available nitrogen at 1.62 mg/kg, available phosphorus at mg/kg, and available potassium at 106 mg/kg. The pH measurement results of the 30 –60 cm soil samples were 8.15, with water-soluble salts at 6.00 g/kg, available nitrogen at 0.82 mg/kg, available phosphorus at 0.09 mg/kg, and available potassium at 66 mg/kg.

Powdered samples (1.000 g) were ultrasonically extracted with 5 mL 100% methanol at 25°C for 60 min (1000 W), followed by centrifugation at 12,000 rpm (4°C, 5 min). Supernatants were collected, and residues were re-extracted with 5 mL methanol. Combined supernatants were diluted to volume in a volumetric flask with methanol. Aliquots (2.5 mL) were syringe-filtered through 0.22 µm membranes for phytochemical analysis. Standard solutions (1.0 mg/mL) were serially diluted in methanol to construct calibration curves (1 – 100 ng/mL). Quantitative analysis was performed using an ACQUITY UPLC H-Class system (Waters) coupled to a Xevo TQS triple quadrupole mass spectrometer. Separation was achieved on an ACQUITY UPLC BEH C18 column (50 × 2.1 mm, 1.7 μm; Waters) maintained at 40°C with a 0.3 mL/min flow rate. The mobile phase consisted of (A) 0.1% formic acid in water and (B) acetonitrile, using the following gradient: 0–2 min 5% B, 2–8 min 5-95% B, 8–10 min 95% B, 10-10.1 min 95-5% B, 10.1–12 min 5% B.

For total flavonoid quantification, 2.00 g of licorice root powder was accurately weighed, wrapped in filter paper, and subjected to Soxhlet extraction with ethanol under 80°C water bath reflux until colorless effluent was observed. The extract was concentrated under reduced pressure, transferred to a 100-mL volumetric flask, and diluted to volume with ethanol. A 0.5-mL aliquot of this solution was mixed with 2.5 mL distilled water in a 10-mL volumetric flask. Sequentially, 0.5 mL of 5% NaNO₂ and 0.5 mL of 10% Al(NO₃)₃ were added with vortex mixing after each addition. After 5-min incubation at room temperature, 2.5 mL of 5% NaOH was added and mixed thoroughly. Following additional 5-min incubation, the solution was diluted to volume with distilled water. Absorbance was measured at 520 nm against a reagent blank using UV-Vis spectrophotometer.

Fresh leaf samples (0.5 g) were homogenized in 4.5 mL ice-cold 50 mM phosphate buffer (pH 7.0 - 7.4) containing 0.1 mM EDTA and 2 mM DTT. The homogenate was centrifuged at 4,000 ×g for 50 min at 4°C, and the supernatant was retained for antioxidant enzyme assays. Content of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), free proline, and malondialdehyde (MDA) were quantified in G. inflata roots using commercial assay kits (Nanjing Jiancheng Bioengineering Institute, China) according to manufacturer protocols.

Fresh tissue samples (0.1 g) were finely chopped and transferred to 50-mL conical flasks containing 10 mL of 95% (v/v) ethanol. After 3 – 5 h of dark incubation at room temperature until complete decolorization occurred, extracts were filtered into 25-mL amber volumetric flasks. The mortar and pestle were rinsed three times with small volumes of 95% ethanol, with washings filtered into the same flask. The combined filtrate was diluted to volume with ethanol and homogenized. Extract aliquots were transferred to cuvettes against a 95% ethanol blank. Absorbance was measured at 665 nm and 649 nm using UV-Vis spectrophotometer.

Total RNA was isolated using the Polyphenolics-Polysaccharides RNA Kit (TIANGEN Biotech, China). cDNA library construction was performed by Majorbio Bio-Pharm Technology Co., Ltd. Poly(A)+ mRNA was enriched from qualified RNA samples using oligo(dT)-attached magnetic beads. Following fragmentation (~300 bp fragments compatible with Illumina HiSeq 6000 platform requirements), first-strand cDNA synthesis was conducted with reverse transcriptase. Second-strand cDNA synthesis generated stable double-stranded DNA, which underwent end repair, adapter ligation, and PCR amplification for library enrichment. Thirty-six cDNA libraries were constructed, representing four G. inflata accessions under salt stress across two timepoints with triplicate biological replicates. Libraries were sequenced on the Illumina HiSeq 6000 platform (150-bp paired-end). Raw reads underwent quality control using SeqPrep and Sickle (Chen et al., 2025) to obtain clean data. High-quality reads were aligned to the G. uralensis reference genome (v2.0) using TopHat2 (Kim et al., 2013)and HISAT2 (Kim et al., 2019).Gene expression quantification was performed with RSEM (Li and Dewey, 2011). Differential expression analysis employed DEGseq (Wang et al., 2010)and DESeq2 (Love et al., 2014) (|log₂FC| > 1, FDR < 0.05). Functional annotation utilized DIAMOND for GO terms and KEGG Orthology (KO) assignment. Enrichment analyses were conducted with GOATools (Klopfenstein et al., 2018)and KOBAS (Bu et al., 2021) (FDR < 0.05). Weighted Gene Co-expression Network Analysis (WGCNA) (Liu et al., 2019)was implemented in R v4.2.2 to identify salt-responsive genes associated with specialized metabolism. An adjacency matrix was constructed using soft thresholding (power = β = 14). Modules were detected via dynamic tree cutting (mergeCutHeight = 0.25, minModuleSize = 30). Module eigengenes (first principal components) represented expression profiles. Spearman correlations between module eigengenes and flavonoid abundances were visualized using ggplot2.

To validate RNA-seq accuracy, six differentially expressed genes (DEGs) regulating flavonoid biosynthesis were selected for qRT-PCR verification( Supplementary Figure S3 ). Specific primers were designed to measure relative expression levels of these genes in root tissues of four G. inflata accessions after 6-h salt treatment. The Glycyrrhiza uralensis lectin gene (Glyur000100s00008376) served as the endogenous reference. Reactions utilized the FastStart Universal SYBR Green Master Mix (Roche) on an Applied Biosystems 7500 Real-Time PCR System (Monad Biotech, China). Relative transcript abundance was calculated via the 2^–ΔΔCt method (Livak and Schmittgen, 2001), Statistical significance was determined by one-way ANOVA with Tukey’s post-hoc test (p < 0.05).

All statistical analyses were performed using R packages and SPSS 27.0. Intergroup differences were assessed by one-way analysis of variance (ANOVA), with post-hoc pairwise comparisons conducted via the least significant difference (LSD) test at a significance threshold of P < 0.05.

Confronting the dual challenges of diminishing wild Glycyrrhiza inflata resources and declining cultivated quality, we evaluated 29 germplasms from the Tarim Basin periphery (coordinates in Supplementary Table S1 ), revealing significant inter-accession variation in bioactive compounds ( Table 1 ) and salt tolerance, as shown in Figure 1 ,all accessions achieved >45% germination in saline-alkali soil (pH 8.15; mean 65.13% ± 8.24%), with 20 exceeding 60% (nine >70%, two >80%, #10 peaking at 87.1%), while plant heights ranged 1.74 – 3.24 m (mean 2.23 m). Critical metabolites varied substantially: total flavonoids (17.93 ± 2.81 to 28.97 ± 5.53 mg/g), glycyrrhizic acid (2.06 ± 0.23 to 7.51 ± 1.48 mg/g), liquiritin (0.72 ± 0.14 to 4.01 ± 1.23 mg/g), liquiritigenin (0.05 ± 0.01 to 0.22 ± 0.03 mg/g), and isoliquiritigenin (0.09 ± 0.01 to 0.29 ± 0.01 mg/g), with hyper-arid desert margin accessions (#1,9,15) showing superior accumulation (P<0.05), suggesting strategic salt stress enhances medicinal biosynthesis. Through triaxial selection ranking germplasms by glycyrrhizic acid content, liquiritin content, and germination rate ( Supplementary Figure S1 )—driven by dual-purpose application in remediating southern Xinjiang’s saline-alkali deserts while generating local economic returns via harvestable bioactive compounds—we prioritized accessions meeting critical germination thresholds due to severely compromised viability in extreme salinity-alkalinity soils, thus selecting: stress-tolerant accessions (#10=T1, #28=T2) with high germination and enriched bioactive compounds, and sensitive accessions (#22=S1, #13=S2) exhibiting moderate germination rates (relatively lower than T1/T2) yet divergent bioactive compound accumulation for comparative stress-response analysis. their controlled-environment metabolite profiles ( Figure 2 ) indicated no significant differences in general flavonoids, or isoglycyrrhizin (P>0.05), but glabridin, licochalcone A,liquiritigenin, liquiritin, and glycyrrhizic acid, The contents of these five active ingredients have varied.

To elucidate salt adaptation mechanisms and select elite germplasms with enhanced stress resilience and bioactive compound production, we first assessed germination rates under varying NaCl concentrations ( Supplementary Figure S2 ). After 15-day exposure to 2% NaCl, significant inter-germplasm divergence emerged: T1 (62%) > T2 (32%) > S2 (8%) > S1 (2%). When plants reached a height of 20 cm, salt stress was applied using a concentration of 150 mM NaCl, previously determined as optimal in preliminary laboratory screening. Root tissues (sampled below the root-shoot junction) were collected at 0, 2, and 6 hours after stress initiation. All samples were immediately washed with distilled water, surface-dried, flash-frozen in liquid nitrogen, and stored at -80°C, with three biological replicates per treatment. As shown in Figure 3 , the control groups of the four Glycyrrhiza inflata varieties exhibited no significant differences in malondialdehyde (MDA), proline, or superoxide dismutase (SOD) levels. However, following salt stress, the tolerant varieties (T1, T2) accumulated significantly higher proline levels ( Figure 3C ) than the sensitive varieties (S1, S2). Conversely, MDA content ( Figure 3E ) was significantly lower in T1 and T2 compared to S1 and S2. Catalase (CAT) activity ( Figure 3A ) was consistently higher in T1 and T2 both before and after stress exposure. These differences likely contribute to the enhanced stress tolerance of T1 and T2.While chlorophyll content ( Figure 3B ) differed between tolerant and sensitive varieties, the absolute difference was relatively small compared to the other parameters (CAT, proline, SOD, MDA) and did not reach statistical significance. Interestingly, SOD activity ( Figure 3D ) in the sensitive varieties (S1, S2) was significantly higher than in the tolerant varieties (T1, T2) after stress and increased progressively with prolonged stress duration. This result contradicts our initial expectations, and the underlying mechanism remains unclear, representing a key question for future investigation.

Overall, the four Glycyrrhiza inflata varieties exhibited distinct baseline levels of the measured compounds even under non-stressed conditions, and employed divergent strategies in response to salt stress. As a key hormone in plant stress responses, abscisic acid (ABA) levels were highest in the tolerant variety T1 under control conditions and remained relatively stable after stress onset. In contrast, the other tolerant variety (T2) significantly upregulated ABA synthesis following stress exposure. Interestingly, the sensitive variety S2 also adopted this strategy of post-stress ABA induction. Conversely, ABA content in the sensitive variety S1 steadily declined after stress imposition ( Figure 4A ), an unexpected pattern that may be a key factor contributing to its stress sensitivity. Regarding indole-3-acetic acid (IAA), post-stress dynamics differed markedly between tolerant and sensitive varieties ( Figure 4B ). The tolerant varieties T1 and T2 exhibited a progressive downregulation of IAA content with prolonged stress duration. Conversely, the sensitive varieties S1 and S2 displayed an increase in IAA levels over the same period. In contrast to both ABA and IAA, the four varieties displayed a consistent pattern for zeatin (ZT) synthesis ( Figure 4C ). All varieties initially increased ZT levels shortly after stress initiation, followed by a subsequent decline as stress duration extended.

Overall, short-term salt stress significantly increased the content of major active compounds in all four Glycyrrhiza inflata varieties, although accumulation patterns differed substantially among varieties for the same compound ( Figure 5 ).As shown in Figures 5A, D , the sensitive varieties (S1, S2) accumulated significantly higher levels of glabridin and licochalcone A at 0, 2, and 6 hours post-stress compared to the tolerant varieties (T1, T2). Conversely, the tolerant varieties (T1, T2) exhibited greater accumulation of glycyrrhizic acid and liquiritin following stress exposure Figures 5B, E ). Notably, glycyrrhizic acid and liquiritin are two primary compounds used in quality assessment of G. inflata. For isoliquiritigenin Figure 5C ), accumulation patterns were similar between T1 and S2, and between T2 and S1.Analysis of the biosynthetic pathways ( Figure 6 ) indicates that, with the exception of glycyrrhizic acid, the other four active compounds (glabridin, licochalcone A, liquiritin, isoliquiritigenin) are synthesized through multi-step reactions originating from coumaroyl-CoA. Notably, isoliquiritigenin and liquiritin share a common pathway, while glabridin and licochalcone A utilize the same precursor. This shared biosynthetic origin likely explains the observed similarities in their accumulation trends. The distinct accumulation patterns of these active compounds between sensitive and tolerant varieties may contribute to their differential stress tolerance.

To validate our hypothesis and identify key salt-responsive genes in flavonoid biosynthesis, we performed transcriptome sequencing on root tissues of two salt-tolerant (T1, T2) and two salt-sensitive (S1, S2) G. inflata germplasms treated with 150 mM NaCl for 0, 2, and 6 hours. Differential expression analysis compared post-stress (2h/6h) versus control (0h) samples across accessions (T1_2h/T1_0h, T1_6h/T1_0h, T2_2h/T2_0h, T2_6h/T2_0h, S1_2h/S1_0h, S1_6h/S1_0h, S2_2h/S2_0h, S2_6h/S2_0h). Bar plots and Venn diagrams visualized DEG counts and overlaps ( Figures 5A, B ). At 2h post-stress, all accessions showed upregulated DEGs, but by 6h, tolerant (T1/T2) and moderately sensitive (S2) lines exhibited ∼50% reduction in upregulated DEGs ( Figure 5C ). Strikingly, S1 maintained elevated DEG counts, suggesting impaired transcriptional regulation under prolonged stress. Although total DEGs ranged 3,161-5,681 per accession, only 26 genes (0.19% of total DEGs) were commonly deregulated across all four germplasms ( Figure 5D ), indicating germplasm-specific transcriptional reprogramming.

GO enrichment analysis of 2h/0h comparisons revealed significant enrichment (FDR < 0.05) in:Oxidative stress response, Flavonoid biosynthesis (T1:16 genes; T2:19; S1:14; S2:16),Flavonoid metabolism, Naringenin 2-hydroxylase activity, Flavonoid 3’-monooxygenase activity (exclusive to T1/S1 with 4–5 genes),Oxidoreductase activity, Hormone-mediated signaling (T1:122; T2:131; S1:91; S2:108 genes).At 6h/0h, pathways shifted toward: Isoflavonoid metabolism (S1-exclusive: 3 genes), Isoflavonoid biosynthesis, Flavonoid biosynthesis (T1:10; T2:15; S1:11; S2:10 genes),Glucosidase activity, Flavonoid 3’-monooxygenase activity (S1-exclusive: 5 genes),Hormone signaling (T1:99; S1:108; S2:81 genes; absent in T2).This temporal progression indicates transcriptional activation peaked at 2h post-stress, with flavonoid pathway genes and Hormone-mediated signaling showing maximal induction ( Figure 7E ). By 6h, attenuated activation coincided with specialized isoflavonoid pathway engagement, particularly in S1.

To functionally characterize differentially expressed genes (DEGs), we performed KEGG enrichment analysis on 2h/0h and 6h/0h comparisons. The top 20 significantly enriched pathways (FDR < 0.01) shared across treatment groups revealed. At 2h post-stress:Isoflavonoid biosynthesis (ko00943), Phenylalanine metabolism (ko00360),Plant MAPK signaling (ko04016), Nitrogen metabolism (ko00910), Ubiquinone/terpenoid-quinone biosynthesis (ko00130), Phenylpropanoid biosynthesis (ko00940). Notably enriched flavonoid pathways: Flavone and flavonol biosynthesis (ko00944), Flavonoid biosynthesis (ko00941),Isoflavonoid biosynthesis (ko00943), Phenylpropanoid biosynthesis (ko00940). Concurrent enrichment of Plant hormone signal transduction (ko04075) suggests coordinated regulation. The significant enrichment (P <0.04) of multiple flavonoid biosynthesis pathways correlates with observed phytochemical upregulation (Section 3.4), indicating transcriptional reprogramming drives stress-induced metabolite accumulation.

Using correlation-based clustering, we grouped co-expressed genes into modules via hierarchical clustering dendrograms, where branch colors represent distinct expression patterns ( Figure 8A ). WGCNA partitioned G. inflata genes into 68 co-expression modules, with the turquoise module being largest (1,235 genes) and plum smallest (31 genes). Module-trait associations were visualized through:Gene-trait correlation heatmap ( Figure 8B ),Mean connectivity distribution ( Figure 8C ), Scale-free topology fit index ( Figure 8D ). Spearman correlation analysis revealed significant compound-module associations: The greenyellow and black modules have the strongest correlation with Glabridin, the darkgreen module has the strongest correlation with Licochalcone A, the cyan module has the strongest correlation with glycyrrhizic acid module, and the plum1 module has the strongest correlation with Liquiritin. To further understand the relationship between the relevant modules and active components, and to identify key genes, we conducted KEGG annotation analysis on all modules with a coefficient of > 0.7. Genes encoding enzymes for primary bioactive compound synthesis were identified across multiple co-expression modules (darkorange, darkgreen, cyan), including Phenylalanine ammonia-lyase (PAL, GinfChr2G00031740) and Chalcone isomerase (CHI, GinfChr1G00083500; GinfChr1G00083530). Within the black module (138 genes), we detected:Five PAL genes (GinfChr1G00091980, GinfChr2G00031750, GinfChr2G00031760, GinfChr5G00197500, GinfChr1G00120940)/One Chalcone reductase (CHR, GinfChr5G00197280),One 3-Hydroxy-3-methylglutaryl-CoA reductase* (HMGR, GinfChr5G00173040),One Cinnamate 4-hydroxylase* (C4H, GinfChr1G00085290), Two 4-Coumarate: CoA ligases* (4CL, GinfChr4G00082360, GinfChr5G00165360), Four Chalcone synthases (CHS, GinfChr3G00137770, GinfChr4G00071830,GinfChr4G00071850).KEGG annotation revealed significant enrichment (P<0.001) in five functional categories ( Figure 9 ): Metabolism (104 genes), Genetic Information Processing, Environmental Information Processing, Cellular Processes, and Organismal Systems. Metabolic genes predominated, with 24 genes each in secondary metabolite biosynthesis and amino acid metabolism. Crucially, this module contained:Phloridzin synthase (flavonoid pathway),2-Hydroxyisoflavanone dehydratase* (HIDH),2,7,4’-Trihydroxyisoflavanone 4’-O-methyltransferase* (HI4OMT),cis-Zeatin O-glucosyltransferase (cis-ZOG, EC:2.4.1.-). These findings indicate coordinated regulation of flavonoid diversification and phytohormone signaling ( Table 2 ). We propose that salt stress induces crosstalk between hormone pathways (zeatin, gibberellins, ethylene, jasmonate) and specialized metabolism, particularly glabridin biosynthesis. Competitive flux partitioning may occur, where synthesis of certain flavonoids/isoflavonoids potentially downregulates glabridin-associated genes.

Based on WGCNA and transcriptome results, we screened genes associated with active ingredient synthesis from the target modules and enrichment analysis. The accumulation levels of major active ingredients across different time points are presented in Figures 10 and 6 . As shown in Figure 10 , in the T1 Glycyrrhiza inflata germplasm, 18 genes within the glycyrrhizin biosynthetic pathway (e.g., GiHMGS1, GiHMGS2, GiHMGR1, GiHMGR2) exhibited significant upregulation 6 hours post-stress. Combined with the accumulation results of active compounds across different stress durations, this pronounced upregulation likely contributed to T1’s substantially higher glycyrrhizin accumulation at 12 hours compared to other germplasms. While the T2 germplasm also showed upregulation of most genes in this pathway, the magnitude of expression change was less pronounced than in T1. In contrast, the stress-sensitive germplasms S1 and S2 reached peak expression levels for most glycyrrhizin pathway genes at 2 hours post-stress, followed by a decline at 6 hours. This expression pattern corresponds to their lower glycyrrhizin accumulation at 6 and 12 hours relative to T1 and T2.Conversely, within the Phenylpropanoid Metabolism Pathway (PMP), the T1 germplasm showed significant downregulation of gene expression at 2 and 6 hours post-stress ( Figure 6 ), exhibiting an expression pattern opposite to that observed in the glycyrrhizin pathway. However, its constitutive (control) expression levels for PMP genes were notably higher than those in the other three germplasms. This elevated baseline likely explains T1’s sustained higher levels of flavonoid and isoflavonoid active ingredients during short-term stress, consistent with previous findings. Germplasms T2, S1, and S2 exhibited upregulation of PMP pathway genes at 2 and 6 hours post-stress, suggesting resource allocation towards synthesizing active compounds like Liquiritin, Licochalcone A, and Glabridin. Notably, S1 displayed higher expression of GiCHI1, GiCHI4, GiIFS1, and GiGT1 at 6 hours compared to the other germplasms, resulting in significantly greater Glabridin synthesis post-stress. T2, meanwhile, upregulated genes such as GiCHS3, GiCHS4, GiCHS5, GiCHR1, and GiCHR2, potentially facilitating increased Isoliquiritigenin and subsequent Liquiritin production.

This study revealed that all 29 tested accessions of Glycyrrhiza inflata exhibited germination rates exceeding 45% under saline-alkaline stress (pH 8.15), with a mean germination rate of 65.13%. Notably, several accessions achieved rates surpassing 80%. Plant height ranged from 1.74 m to 3.24 m, collectively demonstrating the inherent capability of these diverse G. inflata germplasms from the Tarim Basin periphery to thrive in desert saline-alkali soils. Furthermore, the accessions displayed substantial variation in key bioactive constituents: total flavonoid content ranged from 17.926 ± 2.810 mg/g to 28.966 ± 5.533 mg/g, glycyrrhizic acid content from 2.056 ± 0.231 mg/g to 7.506 ± 1.480 mg/g, and liquiritin content from 0.717 ± 0.139 mg/g to 4.008 ± 1.232 mg/g. These results, particularly the high levels of the primary quality indicators glycyrrhizic acid and liquiritin, highlight.

The primary bioactive constituents of Glycyrrhiza inflata are triterpenoids and flavonoids (Yang et al., 2017), These compounds not only exhibit free radical scavenging capacity and antioxidant activity (Kumar and Pandey, 2013), but also function as plant growth regulators (Kumar and Pandey, 2013), playing crucial roles in plant adaptation to environmental conditions and defense against abiotic stress (Shen et al., 2022), Furthermore, studies in both model (Ma et al., 2019) and non-model organisms (Zhang et al., 2023) have demonstrated that appropriate salt stress can effectively enhance desirable traits in cultivated germplasm. Consequently, we subjected roots of four G. inflata germplasms to hydroponic treatment with 150 mM NaCl (the previously determined optimal concentration in our laboratory) for 0h,2h, 6h. Subsequently, we measured changes in physiological parameters, endogenous hormone levels, and key bioactive compound concentrations. Following salt stress exposure, all four germplasms exhibited a characteristic triphasic response in CAT, SOD, and POD enzyme activities: an initial increase, followed by a decline, and then a secondary rise, ultimately stabilizing at levels slightly above or comparable to the control group ( Figure 3 ). Generally, the salt-tolerant germplasms (T1, T2) displayed higher POD, and CAT activities and lower MDA content across most time points compared to the salt-sensitive germplasms (S1, S2). Chlorophyll and free proline content showed no significant changes post-stress, consistent with experimental expectations. This dynamic pattern aligns with findings reported by Ma et al. (Ma et al., 2025b)and Zhang et al(Zhang et al., 2023 ).Concurrently, endogenous hormone levels in all four germplasms underwent varying degrees of fluctuation, representing a physiological effort to mitigate the detrimental effects of salt stress on plant growth. Changes in abscisic acid (ABA), indole-3-acetic acid (IAA), and zeatin (ZT) levels are shown in Figures 4A–C . Notably, S1 displayed a significant decrease in ABA post-stress contrary to its established role as a key stress-responsive hormone in plants (Baron et al., 2012) which may contribute to its lower stress resistance. Salt stress typically triggers resource reallocation from growth to defense mechanisms, reducing auxin (IAA) demand and synthesis (Aizaz et al., 2024). Consistent with this, tolerant germplasms T1 and T2 showed significant IAA downregulation at 2h and 6h post-stress. In contrast, sensitive types S1 and S2 exhibited initial IAA increases; this delayed or attenuated stress response likely contributes to their reduced tolerance. Similarly, ZT levels in T1 remained stable throughout the 6h stress period, mirroring its ABA and CAT response patterns. T2, S1, and S2 displayed significant ZT increases at 2h followed by declines at 6h, representing an adaptive effort to mitigate stress impact. Numerous studies confirm that plants enhance flavonoid synthesis under salt stress to counteract its effects (Xu et al., 2020; Feng et al., 2023), establishing controlled salt exposure as an effective strategy for boosting bioactive compound production. As shown in Figure 5 , all four germplasms exhibited significant upregulation of key bioactive compounds post-stress. Intriguingly, under control conditions, one-year-old plants from all four germplasms—particularly T1, T2, and S2—showed no significant differences in major bioactive compound levels ( Figure 2 ). However, root exposure to 150 mM NaCl induced distinct accumulation patterns: T1/T2 accumulated substantially higher Glycyrrhizic acid than S1/S2; S1/S2 accumulated substantially higher Glabridin and LicochalconeA than T1/T2; Integrating these findings with preceding results, we infer that differential stress-induced expression of genes within flavonoid and glycyrrhizin biosynthetic pathways across the four germplasms underlies these compound-specific accumulation patterns.

Transcriptome analysis in this study revealed substantial variation in the number of differentially expressed genes (DEGs) across different treatment combinations ( Figure 7 ). The T2_6h/T2_0h comparison exhibited the highest number of upregulated genes (3,093, representing 6.43% of expressed genes), alongside 2,567 downregulated genes (5.96%). Conversely, the S2_6h/S2_0h comparison showed relatively fewer DEGs. Overall, most treatment groups induced significant transcriptional reprogramming, characterized by substantial numbers of both upregulated and downregulated genes. This reflects the complexity and diversity of gene expression regulation in different Glycyrrhiza inflata germplasms as they adapt physiologically to varying environmental or experimental conditions. Notably, the number of DEGs at 2h post-stress was slightly higher than at 6h.Gene Ontology (GO) enrichment analysis ( Figure 11 ) indicated that G. inflata orchestrates a multifaceted response to salt stress by differentially regulating genes involved in diverse functions. These include, but are not limited to, flavonoid biosynthesis, modulation of oxidoreductase activity, plant hormone signal transduction, and amino acid degradation. Consistent with the DEG count data, GO terms were enriched with a greater number of genes at 2h compared to 6h, suggesting a more extensive transcriptional response during the initial phase of stress acclimation. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment ( Figure 12 ) further highlighted significant differential expression of genes within key metabolic pathways post-stress, notably flavonoid biosynthesis, flavone and flavonol biosynthesis, and phenylalanine metabolism. The number of differentially expressed genes within these pathways varied considerably among the germplasms, potentially contributing to the observed differences in bioactive compound accumulation.

Recent years have witnessed growing research interest in the clinical efficacy (Liu et al., 2025) therapeutic applications (Liu et al., 2021), and cosmetic utilization (Jiang et al., 2015) of bioactive compounds derived from licorice (Glycyrrhiza spp.). However, the specific biosynthetic mechanisms governing key active constituents, such as flavonoids and glycyrrhizin, in Glycyrrhiza inflata remain incompletely elucidated (Zhao et al., 2023). Furthermore, cultivated licorice, the primary supply source, faces challenges of declining quality, which constrains the development of related industries. Advances in bioinformatics have positioned Weighted Gene Co-expression Network Analysis (WGCNA) as a powerful tool for identifying target genes and key transcription factors (TFs) from RNA-seq data (He et al., 2023).In this study, we employed transcriptome analysis coupled with WGCNA to identify genes associated with bioactive compound accumulation in G. inflata under salt stress and constructed a putative biosynthetic network. Intriguingly, we identified a module (Black) exhibiting a strong negative correlation with Glabridin accumulation. Surprisingly, this module encompassed not only TFs involved in plant hormone signaling pathways (e.g., JAZ, ERF1, PIF3) but also the glycyrrhizin pathway rate-limiting enzyme HMGR and numerous genes encoding enzymes in flavonoid biosynthesis (PAL, 4CL, CHS, CHR).Glycyrrhizin biosynthesis primarily occurs via the mevalonate (MVA) pathway. Within this pathway, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), a key rate-limiting enzyme, catalyzes the conversion of HMG-CoA to mevalonate (MVA), leading to the formation of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) ( Figure 10 ). Conversely, other flavonoid bioactive compounds originate from phenylalanine. Sequential catalysis by phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), and 4-coumarate-CoA ligase (4CL) yields coumaroyl-CoA. This precursor is then channeled through various chalcone synthases (CHS) to synthesize distinct compounds like Glabridin, Liquiritin, and Licochalcone A ( Figure 6 ).Current understanding suggests no direct regulatory interplay between these two pathways. Therefore, our WGCNA finding of a strong negative correlation between HMGR (a key enzyme in glycyrrhizin synthesis) and Glabridin accumulation was unexpected. However, integrating this result with phenotypic data ( Figures 5A, B ) revealed a compelling pattern: the T1 germplasm exhibited the highest glycyrrhizic acid accumulation post-stress but the lowest Glabridin levels among the four germplasms. Conversely, the S1 germplasm accumulated the highest Glabridin but showed no significant change in glycyrrhizic acid within the first 12h post-stress. This inverse relationship led us to hypothesize a potential regulatory mechanism linking these seemingly independent pathways, with HMGR potentially playing a pivotal role. Supporting this hypothesis, transcriptomic data revealed that in the T1 germplasm, genes within the glycyrrhizin pathway (including HMGR) were highly upregulated at 6h post-stress, significantly exceeding levels in other germplasms. Conversely, several genes in the Glabridin pathway (PAL, C4H, 4CL) were downregulated. This coordinated differential expression across multiple genes in both pathways provides corroborative evidence for potential cross-talk. Furthermore, the Glabridin-correlated Black module contained CHR genes, which catalyze the formation of isoliquiritigenin. Notably, isoliquiritigenin, Liquiritin, Glabridin, and Licochalcone A all share coumaroyl-CoA as a common precursor. Their divergence hinges on the specific CHS isoform involved: CHS enzymes producing naringenin chalcone lead to isoliquiritigenin and Liquiritin, while those producing chalcone lead to Glabridin and Licochalcone A. Consequently, we infer that specific CHS isoforms (e.g., GiCHS4, GiCHS5, GiCHS6, which were highly negatively correlated with Glabridin) may preferentially channel coumaroyl-CoA towards alternative flavonoid branches, thereby influencing Glabridin flux. Additionally, the module contained annotated TFs such as ERF1, JAZ, and MYC. ERF1, established in Arabidopsis thaliana, integrates environmental signals to promote local auxin (IAA) accumulation (Li et al., 2021). Elevated IAA typically diverts resources away from stress-related compound synthesis. JAZ proteins act as repressors in jasmonate (JA) signaling; their high expression dampens JA responses (He et al., 2023). Given that JA signaling enhances flavonoid accumulation in plants like tomato (Li et al., 2021), the presence of these TFs within the network further validates the biological relevance of our WGCNA findings.