Morphological, transcriptomic and metabolomic analyses of Sophora davidii mutants for plant height.

Sophora davidii is an important plant resource in the karst region of Southwest China, but S. davidii plant-height mutants are rarely reported. Therefore, we performed phenotypic, anatomic structural, transcriptomic and metabolomic analyses to study the mechanisms responsible for S. davidii plant-height mutants. Phenotypic and anatomical observations showed that compared to the wild type, the dwarf mutant displayed a significant decrease in plant height, while the tall mutant displayed a significant increase in plant height. The dwarf mutant cells were smaller and more densely arranged, while those of the wild type and the tall mutant were larger and loosely arranged. Transcriptomic analysis revealed that differentially expressed genes (DEGs) involved in cell wall biosynthesis, expansion, phytohormone biosynthesis, signal transduction pathways, flavonoid biosynthesis and phenylpropanoid biosynthesis were significantly enriched in the S. davidii plant-height mutants. Metabolomic analysis revealed 57 significantly differential metabolites screened from both the dwarf and tall mutants. A total of 8 significantly different flavonoid compounds were annotated to LIPID MAPS, and three metabolites (chlorogenic acid, kaempferol and scopoletin) were involved in phenylpropanoid biosynthesis and flavonoid biosynthesis. These results shed light on the molecular mechanisms of plant height in S. davidii mutants and provide insight for further molecular breeding programs.

Sophoraflavanone G from Sophora flavescens Ameliorates Allergic Airway Inflammation by Suppressing Th2 Response and Oxidative Stress in a Murine Asthma Model.

Sophoraflavanone G (SG), isolated from Sophora flavescens, has anti-inflammatory and anti-tumor bioactive properties. We previously showed that SG promotes apoptosis in human breast cancer cells and leukemia cells and reduces the inflammatory response in lipopolysaccharide-stimulated macrophages. We investigated whether SG attenuates airway hyper-responsiveness (AHR) and airway inflammation in asthmatic mice. We also assessed its effects on the anti-inflammatory response in human tracheal epithelial cells. Female BALB/c mice were sensitized with ovalbumin, and asthmatic mice were treated with SG by intraperitoneal injection. We also exposed human bronchial epithelial BEAS-2B cells to different concentrations of SG to evaluate its effects on inflammatory cytokine levels. SG treatment significantly reduced AHR, eosinophil infiltration, goblet cell hyperplasia, and airway inflammation in the lungs of asthmatic mice. In the lungs of ovalbumin-sensitized mice, SG significantly promoted superoxide dismutase and glutathione expression and attenuated malondialdehyde levels. SG also suppressed levels of Th2 cytokines and chemokines in lung and bronchoalveolar lavage samples. In addition, we confirmed that SG decreased pro-inflammatory cytokine, chemokine, and eotaxin expression in inflammatory BEAS-2B cells. Taken together, our data demonstrate that SG shows potential as an immunomodulator that can improve asthma symptoms by decreasing airway-inflammation-related oxidative stress.

Integrated transcriptome and small RNA sequencing analyses reveal a drought stress response network in Sophora tonkinensis.

BACKGROUND: Sophora tonkinensis Gagnep is a traditional Chinese medical plant that is mainly cultivated in southern China. Drought stress is one of the major abiotic stresses that negatively impacts S. tonkinensis growth. However, the molecular mechanisms governing the responses to drought stress in S. tonkinensis at the transcriptional and posttranscriptional levels are not well understood. RESULTS: To identify genes and miRNAs involved in drought stress responses in S. tonkinensis, both mRNA and small RNA sequencing was performed in root samples under control, mild drought, and severe drought conditions. mRNA sequencing revealed 66,476 unigenes, and the differentially expressed unigenes (DEGs) were associated with several key pathways, including phenylpropanoid biosynthesis, sugar metabolism, and quinolizidine alkaloid biosynthesis pathways. A total of 10 and 30 transcription factors (TFs) were identified among the DEGs under mild and severe drought stress, respectively. Moreover, small RNA sequencing revealed a total of 368 miRNAs, including 255 known miRNAs and 113 novel miRNAs. The differentially expressed miRNAs and their target genes were involved in the regulation of plant hormone signal transduction, the spliceosome, and ribosomes. Analysis of the regulatory network involved in the response to drought stress revealed 37 differentially expressed miRNA-mRNA pairs. CONCLUSION: This is the first study to simultaneously profile the expression patterns of mRNAs and miRNAs on a genome-wide scale to elucidate the molecular mechanisms of the drought stress responses of S. tonkinensis. Our results suggest that S. tonkinensis implements diverse mechanisms to modulate its responses to drought stress.

Screening and identification of salt-tolerance genes in Sophora alopecuroides and functional verification of SaAQP.

MAIN CONCLUSION: Overexpression of SaAQP can improve the salt tolerance of transgenic soybean hairy roots and A. thaliana. Salt stress severely affects crop yield and food security. There is a need to improve the salt tolerance of crops, but the discovery and utilization of salt-tolerance genes remains limited. Owing to its strong stress tolerance, Sophora alopecuroides is ideal for the identification of salt-tolerance genes. Therefore, we aimed to screen and identify the salt-tolerance genes in S. alopecuroides. With a yeast expression library of seedlings, salt-tolerant genes were screened using a salt-containing medium to simulate salt stress. By combining salt-treatment screening and transcriptome sequencing, 11 candidate genes related to salt tolerance were identified, including genes for peroxidase, inositol methyltransferase, aquaporin, cysteine synthase, pectinesterase, and WRKY. The expression dynamics of candidate genes were analyzed after salt treatment of S. alopecuroides, and salt tolerance was verified in yeast BY4743. The candidate genes participated in the salt-stress response in S. alopecuroides, and their overexpression significantly improved the salt tolerance of yeast. Salt tolerance mediated by SaAQP was further verified in soybean hairy roots and Arabidopsis thaliana, and it was found that SaAQP might enhance the salt tolerance of A. thaliana by participating in a reactive oxygen species scavenging mechanism. This result provides new genetic resources in plant breeding for salt resistance.

Combined Transcriptomic and Metabolomic Analysis Reveals the Role of Phenylpropanoid Biosynthesis Pathway in the Salt Tolerance Process of Sophora alopecuroides.

Salt stress is the main abiotic stress that limits crop yield and agricultural development. Therefore, it is imperative to study the effects of salt stress on plants and the mechanisms through which plants respond to salt stress. In this study, we used transcriptomics and metabolomics to explore the effects of salt stress on Sophora alopecuroides. We found that salt stress incurred significant gene expression and metabolite changes at 0, 4, 24, 48, and 72 h. The integrated transcriptomic and metabolomic analysis revealed that the differentially expressed genes (DEGs) and differential metabolites (DMs) obtained in the phenylpropanoid biosynthesis pathway were significantly correlated under salt stress. Of these, 28 DEGs and seven DMs were involved in lignin synthesis and 23 DEGs and seven DMs were involved in flavonoid synthesis. Under salt stress, the expression of genes and metabolites related to lignin and flavonoid synthesis changed significantly. Lignin and flavonoids may participate in the removal of reactive oxygen species (ROS) in the root tissue of S. alopecuroides and reduced the damage caused under salt stress. Our research provides new ideas and genetic resources to study the mechanism of plant responses to salt stress and further improve the salt tolerance of plants.

Analysis of Phytohormone Signal Transduction in Sophora alopecuroides under Salt Stress.

Salt stress seriously restricts crop yield and quality, leading to an urgent need to understand its effects on plants and the mechanism of plant responses. Although phytohormones are crucial for plant responses to salt stress, the role of phytohormone signal transduction in the salt stress responses of stress-resistant species such as Sophora alopecuroides has not been reported. Herein, we combined transcriptome and metabolome analyses to evaluate expression changes of key genes and metabolites associated with plant hormone signal transduction in S. alopecuroides roots under salt stress for 0 h to 72 h. Auxin, cytokinin, brassinosteroid, and gibberellin signals were predominantly involved in regulating S. alopecuroides growth and recovery under salt stress. Ethylene and jasmonic acid signals may negatively regulate the response of S. alopecuroides to salt stress. Abscisic acid and salicylic acid are significantly upregulated under salt stress, and their signals may positively regulate the plant response to salt stress. Additionally, salicylic acid (SA) might regulate the balance between plant growth and resistance by preventing reduction in growth-promoting hormones and maintaining high levels of abscisic acid (ABA). This study provides insight into the mechanism of salt stress response in S. alopecuroides and the corresponding role of plant hormones, which is beneficial for crop resistance breeding.

Davidones F and G, Two Novel Flavonoids from Sophora davidii (Franch.) Skeels.

An unprecedented novel flavanone davidone F (1) with a seven-membered ring side chain, and a novel flavanonol davidone G (2), along with 11 known flavonoids, were isolated from the ethyl acetate fraction of Sophora davidii (Franch.) Skeels. Their planar structures were established by UV, IR, HRESIMS, 1D and 2D NMR data. The relative configurations of 1 and 2 were determined by calculation of NMR chemical shift values, the absolute configuration of 1 and 2 were assigned by comparing their experimental and calculated electronic circular dichroism (ECD) spectra. Moreover, compounds 1-13 were screened for the translocation activity of glucose transporter 4 (GLUT-4), and the fluorescence intensity was increased to the range of 1.56 and 2.79 folds. Compounds 1 and 2 showed moderate GLUT-4 translocation activity with 1.64 and 1.79 folds enhancement, respectively, at a concentration of 20 µg/mL.

A Molecular-Splicing Strategy for Constructing a Near-Infrared Fluorescent Probe for UDP-Glucuronosyltransferase 1A1.

UDP-glucuronosyltransferase 1A1 (UGT1A1) is a vital metabolic enzyme responsible for the clearance of endogenous substances and drugs. Hitherto, the development of fluorescent probes for UGTs was severely restricted due to the poor isoform selectivity and on-off or blue-shifted fluorescence response. Herein, we established a novel "molecular-splicing" strategy to construct a highly selective near-infrared (NIR) fluorescent probe, HHC, for UGT1A1, which exhibited a NIR signal at 720 nm after UGT1A1 metabolism. HHC was then successfully used for the real-time imaging of endogenous UGT1A1 in living cells and animals and to monitor the bile excretion function. In summary, an isoform-specific NIR fluorescent probe has been developed for monitoring UGT1A1 activity in living systems, high-throughput screening of novel UGT1A1 inhibitors and visual evaluation of bile excretion function.

The Synergistic Effect of Co-Treatment of Methyl Jasmonate and Cyclodextrins on Pterocarpan Production in Sophora flavescens Cell Cultures.

Pterocarpans are derivatives of isoflavonoids, found in many species of the family Fabaceae. Sophora flavescens Aiton is a promising traditional Asian medicinal plant. Plant cell suspension cultures represent an excellent source for the production of valuable secondary metabolites. Herein, we found that methyl jasmonate (MJ) elicited the activation of pterocarpan biosynthetic genes in cell suspension cultures of S. flavescens and enhanced the accumulation of pterocarpans, producing mainly trifolirhizin, trifolirhizin malonate, and maackiain. MJ application stimulated the expression of structural genes (PAL, C4H, 4CL, CHS, CHR, CHI, IFS, I3'H, and IFR) of the pterocarpan biosynthetic pathway. In addition, the co-treatment of MJ and methyl-ß-cyclodextrin (MeßCD) as a solubilizer exhibited a synergistic effect on the activation of the pterocarpan biosynthetic genes. The maximum level of total pterocarpan production (37.2 mg/g dry weight (DW)) was obtained on day 17 after the application of 50 µM MJ on cells. We also found that the combined treatment of cells for seven days with MJ and MeßCD synergistically induced the pterocarpan production (trifolirhizin, trifolirhizin malonate, and maackiain) in the cells (58 mg/g DW) and culture medium (222.7 mg/L). Noteworthy, the co-treatment only stimulated the elevated extracellular production of maackiain in the culture medium, indicating its extracellular secretion; however, its glycosides (trifolirhizin and trifolirhizin malonate) were not detected in any significant amounts in the culture medium. This work provides new strategies for the pterocarpan production in plant cell suspension cultures, and shows MeßCD to be an effective solubilizer for the extracellular production of maackiain in the cell cultures of S. flavescens.

Effects of alkaloids from Sophora flavescens on osteoblasts infected with Staphylococcus aureus and osteoclasts.

Chronic osteomyelitis is primarily caused by infection with Staphylococcus aureus (S. aureus). Antibiotics are commonly administered; however, it is a challenge to promote bone healing. The aim of this study was to investigate the in vitro effects of alkaloids from the herbal remedy Sophora flavescens (ASF) on rat calvarial osteoblasts (ROBs) infected with S. aureus and healthy osteoclasts. Cell proliferation and alkaline phosphatase, interleukin-6, and tumour necrosis factor-α activity was measured in infected ROBs; tartrate-resistant acid phosphatase was evaluated in osteoclasts via enzyme-linked immunosorbent assay. The mRNA and protein expression levels of bone morphogenetic protein 2, runt-related transcription factor 2, osteoprotegerin, and receptor activator of nuclear factor kappa-B ligand were assessed in infected ROBs through reverse transcription-polymerase chain reaction and western blotting analysis, respectively. Results indicated that ASF increased the viability of uninfected ROBs and infected ROBs treated with vancomycin via regulation of bone morphogenetic protein 2, runt-related transcription factor, osteoprotegerin, and receptor activator of nuclear factor kappa-B ligand mRNA and protein expression levels. In addition, the secretion of the inflammatory factor tumour necrosis factor-α was decreased and alkaline phosphatase activity was increased, inhibiting the viability of osteoclasts and tartrate-resistant acid phosphatase activity. Therefore, the herbal remedy ASF has potential as a new treatment for chronic osteomyelitis.

Profiling of the Major Phenolic Compounds and Their Biosynthesis Genes in Sophora flavescens Aiton.

Sophorae Radix (Sophora flavescens Aiton) has long been used in traditional medicine in East Asia due to the various biological activities of its secondary metabolites. Endogenous contents of phenolic compounds (phenolic acid, flavonol, and isoflavone) and the main bioactive compounds of Sophorae Radix were analyzed based on the qualitative HPLC analysis and evaluated in different organs and at different developmental stages. In total, 11 compounds were detected, and the composition of the roots and aerial parts (leaves, stems, and flowers) was significantly different. trans-Cinnamic acid and p-coumaric acid were observed only in the aerial parts. Large amounts of rutin and maackiain were detected in the roots. Four phenolic acid compounds (benzoic acid, caffeic acid, ferulic acid, and chlorogenic acid) and four flavonol compounds (kaempferol, catechin hydrate, epicatechin, and rutin) were higher in aerial parts than in roots. To identify putative genes involved in phenolic compounds biosynthesis, a total of 41 transcripts were investigated. Expression patterns of these selected genes, as well as the multiple isoforms for the genes, varied by organ and developmental stage, implying that they are involved in the biosynthesis of various phenolic compounds both spatially and temporally.

Identification of Genes Involved in Flavonoid Biosynthesis in Sophora japonica Through Transcriptome Sequencing.

Sophora japonica is a traditional Chinese medicinal ingredient that is widely used in the medicine, food, and industrial dye industries. Since flavonoids are the main components of S. japonica, studying the flavonoid composition and content of this plant is important. This study aimed to identify molecules involved in the flavonoid biosynthetic pathways in S. japonica. Deep sequencing was performed, and 85,877,352 clean reads were filtered from 86,095,152 raw reads. The clean reads were spliced to obtain 111,382 unigenes, which were then annotated with NR, GO, KEGG, eggNOG. Differential expression analysis and NR function prediction revealed 18 differentially expressed unigenes associated with 13 enzymes in flavonoid biosynthetic pathways. Our results reveal new insights on secondary metabolite biosynthesis-related genes in S. japonica and enhance the potential applications of S. japonica in genetic engineering.

Impact of Funneliformis mosseae on the growth, lead uptake, and localization of Sophora viciifolia.

On the basis of a pot experiment under lead (Pb) stress, we investigated the effects of an arbuscular mycorrhizal (AM) fungus (Funneliformis mosseae) on the growth and Pb uptake of Sophora viciifolia L., and explored the Pb localization in AM roots using transmission electron microscopy (TEM) and energy dispersive X-ray spectroscopy (EDS). The results showed that high Pb levels (500 and 1000 µg/g) inhibited the growth of S. viciifolia seedlings. Compared with the noninoculation treatment, F. mosseae inoculation decreased the Pb concentrations above- and belowground by 61.0% and 15.2%, when exposed to Pb at a concentration of 1000 µg/g. The root length, fork number, tip number, surface area, and volume of mycorrhizal S. viciifolia were higher than those of the corresponding nonmycorrhizal plants. These parameters of mycorrhizal plants increased by 220%, 219%, 157%, 225%, and 278% when plants were exposed to Pb at 1000 µg/g compared with nonmycorrhizal plants. The ratio of root length with diameters between 0-0.2 mm to the total root length significantly increased under Pb stress, and F. mosseae inoculation significantly reduced the ratio. Under Pb stress, F. mosseae increased the ratios of root length with 0.61-0.8 and 0.81-1.0 mm diameters to the total root length, indicating that F. mosseae tended to thicken the roots of S. viciifolia under Pb additions. The combined results of TEM and EDS indicated that Pb deposited in not only plant cells but also the cell walls and vacuoles of the AM fungal intracellular hyphae, thus revealing the subcellular-level mechanism of AM fungi in alleviating the Pb toxicity to the host plant.

Transcriptome Analysis of Nine Tissues to Discover Genes Involved in the Biosynthesis of Active Ingredients in Sophora flavescens.

Sophora flavescens AITON (kurara) has long been used to treat various diseases. Although several research findings revealed the biosynthetic pathways of its characteristic chemical components as represented by matrine, insufficient analysis of transcriptome data hampered in-depth analysis of the underlying putative genes responsible for the biosynthesis of pharmaceutical chemical components. In this study, more than 200 million fastq format reads were generated by Illumina's next-generation sequencing approach using nine types of tissue from S. flavescens, followed by CLC de novo assembly, ultimately yielding 83,325 contigs in total. By mapping the reads back to the contigs, reads per kilobase of the transcript per million mapped reads values were calculated to demonstrate gene expression levels, and overrepresented gene ontology terms were evaluated using Fisher's exact test. In search of the putative genes relevant to essential metabolic pathways, all 1350 unique enzyme commission numbers were used to map pathways against the Kyoto Encyclopedia of Genes and Genomes. By analyzing expression patterns, we proposed some candidate genes involved in the biosynthesis of isoflavonoids and quinolizidine alkaloids. Adopting RNA-Seq analysis, we obtained substantially credible contigs for downstream work. The preferential expression of the gene for putative lysine/ornithine decarboxylase committed in the initial step of matrine biosynthesis in leaves and stems was confirmed in semi-quantitative polymerase chain reaction (PCR) analysis. The findings in this report may serve as a stepping-stone for further research into this promising medicinal plant.

Flavonoids and arylbenzofurans from the rhizomes and roots of Sophora tonkinensis with IL-6 production inhibitory activity.

Three new compounds (1-3) and 20 known compounds were isolated from the rhizomes and roots of Sophora tonkinensis, and all the isolates were tested for their inhibitory activity against IL-6 production in HMC-1 cells stimulated by PMA plus ionophore, A23187. Of the tested compounds, compounds 1, 5, 9, and 21 were found to potently inhibit IL-6 production with IC50 values of 1.62, 0.73, 3.01, and 4.02 µM, respectively.

Antitumor effects of concanavalin A and Sophora flavescens lectin in vitro and in vivo.

AIM: Proteins with legume lectin domains are known to possess a wide range of biological functions. Here, the antitumor effects of two representative legume lectins, concanavalin A (ConA) and Sophora flavescens lectin (SFL), on human breast carcinoma cells were investigated in vitro and in vivo. METHODS: Human breast carcinoma MCF-7 cells and human normal mammary epithelial MCF-10A cells were examined. Cell viability was detected using WST-1 and CCK-8 assays. Cell apoptosis was analyzed with Hoechst 33258 staining. Cell cycle was investigated using flow cytometry. The expression of relevant proteins was measured using Western blotting. Breast carcinoma MCF-7 bearing nude mice were used to study the antitumor effects in vivo. The mice were injected with ConA (40 mg/kg, ip) and SFL (55 mg/kg, ip) daily for 14 d. RESULTS: ConA and SFL inhibited the growth of MCF-7 cells in dose- and time-dependent manners (IC50 values were 15 and 20 µg/mL, respectively). Both ConA and SFL induced apoptotic morphology in MCF-7 cells without affecting MCF-10A cells. ConA and SFL dose-dependently increased the sub-G1 proportion in MCF-7 cells, while SFL also triggered the G2/M phase cell cycle arrest. Both ConA and SFL dose-dependently increased the activities of caspase-3 and caspase-9 and release of cytochrome C from mitochondria into cytoplasm, up-regulated Bax and Bid, and down-regulated Bcl-2 and Bcl-XL in MCF-7 cells. ConA reduced NF-κB, ERK, and JNK levels, and increased p53 and p21 levels, while SFL caused similar changes in NF-κB, ERK, p53, and p21 levels, but did not affect JNK expression. Administration of ConA and SFL significantly decreased the subcutaneous tumor mass volume and weight in MCF-7 bearing nude mice. CONCLUSION: ConA and SFL exert anti-tumor actions against human breast carcinoma MCF-7 cells both in vitro and in vivo.

Multi-omics analysis uncovers the transcriptional regulatory mechanism of magnesium Ions in the synthesis of active ingredients in Sophora tonkinensis.

Magnesium (Mg) plays a pivotal role as an essential component of plant chlorophyll and functions as a critical coenzyme. However, research exploring the regulatory mechanisms of magnesium ions on the synthesis of secondary metabolites is still in its early stages. Sophora tonkinensis is a widely utilized medicinal plant in China, recognized for its diverse secondary metabolites with active properties. This study investigates variations in these ingredients in tissue-cultured seedlings under varying magnesium concentrations. Simultaneously, an omics data analysis was conducted on tissue-cultured seedlings subjected to treatments with magnesium and low magnesium. These comprehensive omics analyses aimed to elucidate the mechanisms through which magnesium influences active components, growth, and development. Magnesium exerts a pervasive influence on various metabolic pathways, forming an intricate network. Research findings indicate that magnesium impacts diverse metabolic processes, including the absorption of potassium and calcium, as well as photosynthetic activity. Consequently, these influences lead to discernible changes in the levels of pharmacologically active compounds and the growth and developmental status.This study is the first to employ a multi-omics data analysis in S. tonkinensis. This methodology allows us to uncover the overarching impact of metabolic networks on the levels of various active ingredients and specific phenotypes.

Molecular characterization of a membrane-bound prenyltransferase specific for isoflavone from Sophora flavescens.

Prenylated isoflavones are secondary metabolites that are mainly distributed in legume plants. They often possess divergent biological activities such as anti-bacterial, anti-fungal, and anti-oxidant activities and thus attract much attention in food, medicinal, and agricultural research fields. Prenyltransferase is the key enzyme in the biosynthesis of prenylated flavonoids by catalyzing a rate-limiting step, i.e. the coupling process of two major metabolic pathways, the isoprenoid pathway and shikimate/polyketide pathway. However, so far only two genes have been isolated as prenyltransferases involved in the biosynthesis of prenylated flavonoids, namely naringenin 8-dimethylallyltransferase from Sophora flavescens (SfN8DT-1) specific for some limited flavanones and glycinol 4-dimethylallyltransferase from Glycine max (G4DT), specific for pterocarpan substrate. We have in this study isolated two novel genes coding for membrane-bound flavonoid prenyltransferases from S. flavescens, an isoflavone-specific prenyltransferase (SfG6DT) responsible for the prenylation of the genistein at the 6-position and a chalcone-specific prenyltransferase designated as isoliquiritigenin dimethylallyltransferase (SfiLDT). These prenyltransferases were enzymatically characterized using a yeast expression system. Analysis on the substrate specificity of chimeric enzymes between SfN8DT-1 and SfG6DT suggested that the determinant region for the specificity of the flavonoids was the domain neighboring the fifth transmembrane α-helix of the prenyltransferases.

Using ITS2 PCR-RFLP to generate molecular markers for authentication of Sophora flavescens Ait.

BACKGROUND: Dried root of Sophora flavescens Ait. is a medicinal material occasionally misused or adulterated by other species similar in appearance. In this study the internal transcribed spacer (ITS) regions of DNA samples of S. flavescens Ait. collected from different areas of Taiwan were amplified by polymerase chain reaction (PCR) and compared. The effectiveness of using ITS2 PCR restriction fragment length polymorphism (RFLP)-generated markers to differentiate S. flavescens Ait. from possible adulterants was also evaluated. RESULTS: The S. flavescens Ait. samples collected from different areas were extremely low in ITS sequence variability at species level. ITS2 PCR-RFLP coupled with restriction enzymes Sac I, Sac II, Xho I or Pvu I produced specific fragments for all tested variants. ITS2 PCR-RFLP coupled with Sac II was further performed to identify mixtures of DNA extracts of S. flavescens Ait. and Sophora tomentosa L. in various ratios. The developed ITS2 PCR-RFLP markers could detect mixed DNA samples of S. flavescens Ait./S. tomentosa L. up to a ratio of 10:1. CONCLUSION: The present study demonstrates the usefulness of ITS2 PCR-RFLP coupled with pre-selected restriction enzymes for practical and accurate authentication of S. flavescens Ait. The technique is also suitable for analysing S. flavescens Ait. mixed with other adulterants.

TMT based proteomic profiling of Sophora alopecuroides leaves reveal flavonoid biosynthesis processes in response to salt stress.

Salt stress is the major abiotic stress worldwide, adversely affecting crop yield and quality. Utilizing salt tolerance genes for the genetic breeding of crops is one of the most effective measures to withstand salinization. Sophora alopecuroides is a well-known saline-alkaline and drought-tolerant medicinal plant. Understanding the underlying molecular mechanism for Sophora alopecuroides salt tolerance is crucial to identifying the salt-tolerant genes. In this study, we performed tandem mass tag (TMT) based proteomic profiling of S. alopecuroides leaves under 150 mM NaCl induced salt stress condition for 3 d and 7 d. Data are available on ProteomeXchange (PXD027627). Furthermore, the proteomic findings were validated through parallel reaction monitoring (PRM). We observed that the expression levels of several transporter proteins related to the secondary messenger signaling pathway were altered under salt stress conditions induced for 3 d. However, the expression of the certain transferase, oxidoreductase, dehydrogenase, which are involved in the biosynthesis of flavonoids, alkaloids, phenylpropanoids, and amino acid metabolism, were mainly alerted after 7 d post-salt-stress induction. Several potential genes that might be involved in salt stress conditions were identified; however, it demands further investigation. Although salt stress affects the level of secondary metabolites, their correlation needs to be investigated further. SIGNIFICANCE: Salinization is the most severe abiotic adversity, which has had a significant negative effect on world food security over the time. Excavating salt-tolerant genes from halophytes or medicinal plants is one of the important measures to cope with salt stress. S. alopecuroides is a well-known medicinal plant with anti-tumor, anti-inflammatory, and antibacterial effects, anti-saline properties, and resistance to drought stress. Currently, only a few studies have explored the S. alopecuroides' gene function, and regulation and these studies are mostly related to the unpublished genome sequence information of S. alopecuroides. Recently, transcriptomics and metabolomics studies have been carried on the abiotic stress in S. alopecuroides roots. Multiple studies have shown that altered gene expression at the transcript level and altered metabolite levels do not correspond to the altered protein levels. In this study, TMT and PRM based proteomic analyses of S. alopecuroides leaves under salt stress condition induced using 150 mM NaCl for 3 d and 7 d was performed. These analyses elucidated the activation of different mechanisms in response to salt stress. A total of 434 differentially abundant proteins (DAPs) in salt stress conditions were identified and analyzed. For the first time, this study utilized proteomics technology to dig out plentiful underlying salt-tolerant genes from the medicinal plant, S. alopecuroides. We believe that this study will be of great significance to crop genetics and breeding.