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Isatidis Folium , derived from the dried leaves of Isatis indigotica Fort., has been used for centuries as a traditional Chinese herb with antibacterial and antiviral properties. However, heterogeneity in cultivation conditions and climatic variations poses challenges to accurately and effectively evaluate its quality. Current quality control methods cannot provide a comprehensive and effective identification of herbal quality and preparation efficacy. This study aimed to investigate the impact of different environmental factors on the biosynthesis and accumulation of medicinal components and identify biomarker genes and functional proteins associated with abiotic stress responses of  Isatis indigotica  Fort. We proposed evaluating the quality of Isatidis Folium based on multi-component quantitative analysis and integrating transcriptomic, proteomic, and physiological indicators to elucidate the mechanisms of herbal quality variation. The results revealed that abiotic stress conditions significantly altered the levels of bioactive constituents, physiological indices, and specific genes and proteins. Notably, biological pathways such as porphyrin metabolism, photosynthesis, and carbon fixation by photosynthetic organisms were implicated in phototoxicity within the photosystem under abiotic stresses. Biological pathways related to indole metabolism, specifically phenylalanine, tyrosine, and tryptophan synthesis, tryptophan metabolism, and indole alkaloid synthesis, were recognized as critical regulatory networks modulating indole alkaloid content. Candidate biomarkers such as HemB , PsbB , RBS2 , RIBA2 , TRPA , and TRPB were identified as potential factors of quality deterioration under adverse conditions. Based on the integration of chemical analysis and multi-omics techniques, a new hierarchical quality control scenario for Isatidis Folium was finally proposed, providing a research foundation for the innovation-driven development of traditional Chinese medicine.

In this work, Isatis tinctoria hairy root cultures (ITHRCs) were established as an alternative source for flavonoids (FL) production. I. tinctoria hairy root line V was found to be the most efficient line and was further confirmed by the PCR amplification of rolB, rolC and aux1 genes. Culture parameters of ITHRCs were optimized by Box-Behnken design (BBD), and eight bioactive FL constituents (rutin, neohesperidin, buddleoside, liquiritigenin, quercetin, isorhamnetin, kaempferol and isoliquiritigenin) were quali-quantitatively determined by LC-MS/MS. Under optimal conditions, the total FL accumulation of ITHRCs (24 day-old) achieved was 438.10 μg/g dry weight (DW), which exhibited significant superiority as against that of 2 year-old field grown roots (341.73 μg/g DW). Additionally, in vitro antioxidant assays demonstrated that ITHRCs extracts exhibited better antioxidant activities with lower IC₅₀ values (0.41 and 0.39, mg/mL) as compared to those of field grown roots (0.56 and 0.48, mg/mL). To the best of our knowledge, this is the first report describing FL production and antioxidant activities from ITHRCs.

Key messageIiSVP of Isatis indigotica was cloned and its expression pattern was analyzed. Ectopic expression of IiSVP in Arabidopsis could delay the flowering time and reduce the size of the floral organs.SVP (SHORT VEGETATIVE PHASE) can negatively regulate the flowering time of Arabidopsis. In the present work, the cDNA of IiSVP, an orthologous gene of AtSVP in I. indigotica, was cloned. IiSVP was highly expressed in rosette leaves, inflorescences and petals, but weakly expressed in sepals, pistils and young silicles. The results of subcellular localization showed that IiSVP was localized in nucleus. Bioinformatics analysis indicated that this protein was a MADS-box transcription factor. Constitutive expression of IiSVP in Arabidopsis thaliana resulted in decrease of the number of petals and stamens, and curly sepals were formed. In IiSVP transgenic Arabidopsis plants, obvious phenotypic variations in flowers could be observed, especially the size of the floral organs. In comparison with the wild-type plants, the size of petals, stamens and pistil in IiSVP transgenic Arabidopsis plants was decreased significantly. In some transgenic plants, the petals were wrapped by the sepals. Yeast two-hybrid experiments showed that IiSVP could form higher-order complexes with other MADS proteins, including IiSEP1, IiSEP3, IiAP1 and IiSEP4, but could not interact with IiSEP2. In this work, it was proved that the flowering process and the floral development in Arabidopsis could be affected by IiSVP from I. indigotica Fortune.

Superoxide dismutase (SOD) is a key enzyme in the plant antioxidant system. It plays an essential role in plant adversity stress by scavenging excess reactive oxygen species to protect cells from oxidative damage. Isatis indigotica, being a mildly saline-tolerant plant, can be grown in soils containing a certain amount of saline–alkaline content. In order to reveal the SOD gene family members and their potential roles under saline and alkaline stress, the present study used a bioinformatics approach to identify 9 potential IiSOD genes in the I. indigotica genome. It analyzed the expression patterns of SOD family genes (IiSODs) in response to alkaline stress. According to the results of quantitative real-time PCR (qRT-PCR), the expression levels of the IiSOD7 gene significantly increased within 120 h of alkaline stress treatment, while the expression level of the IiSOD8 gene was the highest among all detected genes at 120 h of alkaline stress. The rest of the genes showed different degrees of expression. Alkaline stress showed significant and dynamic changes in the content of indigo and indirubin in leaves of I. indigotica. Finally, the yeast one-hybrid assay confirmed that IiWRKY54 was able to activate the expression of IiSOD2 and IiSOD7. Combined with qRT-PCR analysis, it was further hypothesized that IiWRKY54 might enhance the alkaline tolerance of I. indigotica by regulating the expression of IiSOD2 and IiSOD7. Taken together, this study lays the foundation for elucidating the function of the IiSOD gene in salinity stress tolerance of I. indigotica as well as promoting the genetic breeding of alkaline-tolerant varieties of I. indigotica.

Isatidis Folium (Daqingye, DQY) and Isatidis Radix (Banlangen, BLG) are the leaf and root of the plant Isatis tinctoria Fort. (syn. Isatis indigotica Fort.), commonly prescribed for detoxification, and the inhibition of viral and oxidative activities. Given their widespread use, we set forth to investigate the authenticity and chemical composition of DQY and BLG samples obtained from eighteen administrative districts in the Hong Kong market. The present study screened the identities and chemical composition of DQY and BLG through molecular authentication and HPLC methods, respectively. Molecular authentication utilized DNA barcoding, focusing on nuclear ribosomal and chloroplast regions. The HPLC methods were conducted in accordance with the Hong Kong Chinese Materia Medica Standards (HKCMMS). We found that only one sample was genuine according to the species definition in the Chinese Pharmacopoeia and HKCMMS for both herbs. The chemical composition of the adulterated and the genuine samples were completely different, that the adulterant samples did not have the standard chemical markers epigoitrin and indirubin of Banlangen and Daqingye as listed in the Chinese Pharmacopoeia. Our investigation underscores the widespread substitution by Strobilanthes (Nanbanlangen and Nandaqingye) species, mainly due to the preference of use of this herb in southern China. The adulteration of Daqingye by Blumea balsamifera (Ainaxiang) was probably due to mislabeling in the herb shop, though the error might have originated from the supplying sources. We recommend providing education on the necessity of using authentic Daqingye and Banlangen, especially in combined regimens, to standardize treatment effects. More education is also needed on the morphological differentiation of Banlangen from Nanbanlangen and Daqingye from Nandaqingye. The implementation of a track-and-trace system is strongly recommended to prevent and deter incorrect supply chain practices that lead to substitution or adulteration.

Isatis indigotica is a widely used herb for the clinical treatment of colds, fever, and influenza in Traditional Chinese Medicine (TCM). Various structural classes of compounds have been identified as effective ingredients. However, little is known at genetics level about these active metabolites. In the present study, we performed de novo transcriptome sequencing for the first time to produce a comprehensive dataset of I. indigotica. A database of 36,367 unigenes (average length = 1,115.67 bases) was generated by performing transcriptome sequencing. Based on the gene annotation of the transcriptome, 104 unigenes were identified covering most of the catalytic steps in the general biosynthetic pathways of indole, terpenoid, and phenylpropanoid. Subsequently, the organ-specific expression patterns of the genes involved in these pathways, and their responses to methyl jasmonate (MeJA) induction, were investigated. Metabolites profile of effective phenylpropanoid showed accumulation pattern of secondary metabolites were mostly correlated with the transcription of their biosynthetic genes. According to the analysis of UDP-dependent glycosyltransferases (UGT) family, several flavonoids were indicated to exist in I. indigotica and further identified by metabolic profile using UPLC/Q-TOF. Moreover, applying transcriptome co-expression analysis, nine new, putative UGTs were suggested as flavonol glycosyltransferases and lignan glycosyltransferases. This database provides a pool of candidate genes involved in biosynthesis of effective metabolites in I. indigotica. Furthermore, the comprehensive analysis and characterization of the significant pathways are expected to give a better insight regarding the diversity of chemical composition, synthetic characteristics, and the regulatory mechanism which operate in this medical herb.

Background Plant adaptation to environmental stress is crucial for improving crop resilience and productivity. The growth and yield of Isatis indigotica are significantly affected by water conditions. In this study, high-throughput transcriptome sequencing was performed on leaf samples from Isatis indigotica after different treatments: normal precipitation (CK), 40% rainfall reduction (R1), 80% rainfall reduction (R2), 40% rainfall enhancement (I1) and 80% rainfall enhancement (I2). Results Under 80% rainfall augmentation (I2), the malondialdehyde (MDA) content of Isatis indigotica leaves was the lowest, and the proline (pro) and catalase (CAT) activities were the highest. These findings indicate that normal precipitation conditions do not meet the optimal water requirements for the growth of Isatis indigotica and that appropriate irrigation can be used to improve the accumulation and quality of medicinal substances from this species. Transcriptome analysis of Isatis indigotica leaves compared with those in the control group (CK) revealed 896, 2551, 1294, and 3082 differentially expressed genes in the reduced rainfall reduction groups (R1, R2) and increased rainfall groups (I1, 12), respectively. The number of differentially expressed genes (DEGs) gradually increased with increasing rainfall and decreased after rainfall reduction. The GO enrichment results revealed that the DEGs were significantly enriched in functions such as cellular processes, metabolic processes, stimulus response, cell structure, and catalytic and binding activities. KEGG analysis revealed that metabolic pathways such as glutathione metabolism, phenylpropanoid biosynthesis, and plant hormone signaling were significantly enriched, with the greatest number of enriched genes. This study revealed 32 antioxidant system-related genes, 49 phenylpropanoid biosynthesis-related genes, and 49 plant hormone signaling pathway-related genes among the significantly enriched pathways. Conclusions This study provides new insights into the regulation of Isatis indigotica leaves in response to different water contents at the molecular level. The findings also provide a reference for optimizing the field management of Isatis indigotica and improving the quality and yield of medicinal materials.

Background Isatis indigotica Fort. is one of the most commonly used traditional Chinese medicines. Its antiviral compound is a kind of lignan, which is formed with the action of dirigent proteins (DIR). DIR proteins are members of a large family of proteins which impart stereoselectivity on the phenoxy radical-coupling reaction, yielding optically active lignans from two molecules of E -coniferyl alcohol. They exist in almost every vascular plant. However, the DIR and DIR-like protein gene family in I. indigotica has not been analyzed in detail yet. This study focuses on discovery and analysis of this protein gene family in I. indigotica for the first time . Results Analysis of transcription profiling database from I. indigotica revealed a family of 19 full-length unique DIR and DIR-like proteins. Sequence analysis found that I. indigotica DIR and DIR-like proteins ( Ii DIR) were all-beta strand proteins, with a signal peptide at the N -terminus. Phylogenetic analysis of the 19 proteins indicated that the Ii DIR genes cluster into three distinct subfamilies, DIR-a, DIR-b/d, and DIR-e, of a larger plant DIR and DIR-like gene family. Gene-specific primers were designed for 19 unique Ii DIRs and were used to evaluate patterns of constitutive expression in different organs. It showed that most Ii DIR genes were expressed comparatively higher in roots and flowers than stems and leaves. Conclusions New DIR and DIR-like proteins were discovered from the transcription profiling database of I. indigotica through bioinformatics methods for the first time. Sequence characteristics and transcript abundance of these new genes were analyzed. This study will provide basic data necessary for further studies.

The giant organs and enhanced concentrations of secondary metabolites realized by autopolyploidy are attractive for breeding the respective medicinal and agricultural plants and studying the genetic mechanisms. The traditional medicinal plant Chinese woad (Isatis indigotica Fort., 2n = 2x = 14) is now still largely used for the diseases caused by bacteria and viruses in China. In this study, its autopolyploids (3x, 4x) were produced and characterized together with the 2x donor for their phenotype and transcriptomic alterations by using high-throughput RNA sequencing. With the increase of genome dosage, the giantism in cells and organs was obvious and the photosynthetic rate was higher. The 4x plants showed predominantly the normal meiotic chromosome pairing (bivalents and quadrivalents) and equal segregation and then produced the majority of 4x progeny. The total 70136 All-unigenes were de novo assembled, and 56,482 (80.53%) unigenes were annotated based on BLASTx searches of the public databases. From pair-wise comparisons between transcriptomic data of 2x, 3x, 4x plants, 1856 (2.65%)(2x vs 4x), 693(0.98%)(2x vs 3x), 1045(1.48%)(3x vs 4x) unigenes were detected to differentially expressed genes (DEGs), including both up- and down-regulated ones. These DEGs were mainly involved in cell growth (synthesis of expansin and pectin), cell wall organization, secondary metabolite biosynthesis, response to stress and photosynthetic pathways. The up-regulation of some DEGs for metabolic pathways of functional compounds in the induced autotetraploids substantiates the promising new type of this medicinal plant with the increased biomass and targeted metabolites.

RBOH gene may regulate the resistance of Isatis indigotica Fort. to salt stress by mediating the production of H O . RBOH gene plays an important role in plant growth and development, abiotic and biotic stress response, and hormone signalling. However, studies on RBOH gene expression and molecular mechanism of Isatis indigotica Fort. under salt stress have not been reported. This study identified 10 genes of the I. indigotica RBOH gene family (IiRBOH) and divided them into five subfamilies (I-V). Genes within the same class show conserved structural features and similar amino acid sequences. Analysis of CRE suggested that IiRBOH genes might play roles in growth and development, metabolism, hormone regulation, and stress response. Two physiological indicators of I. indigotica treated with salt for different days were detected. It was found that the content of H O in the I. indigotica tissue first increased, then decreased and increased again. The catalase activity also showed a trend of first increasing and then decreasing. The qRT-PCR results showed that these IiRBOH genes showed different expression patterns in response to salt stress, and some of these genes may be involved in the resistance of I. indigotica to salt stress. Through RT-PCR analysis and screening on the PlantCARE website, it was found that IiRBOHA and IiRBOHC not only possess W-box CRE but also exhibit high expression under salt stress. Y1H experiments were conducted with the WRKY genes predicted by phylogenetic analysis to regulate salt stress potentially, and it was discovered that IiWRKY6 and IiWRKY54 can directly activate the transcription of the IiRBOHA gene promoter. This study preliminarily explored the mechanism by which the RBOH gene in I. indigotica mediates H O to resist salt stress, thus laying a foundation for further research on the biological functions of the RBOH gene in I. indigotica.