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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.

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.

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.

Recovering evidence for the intentional use of plants in the Palaeolithic is challenging due to their perishable nature as, unlike chipped stone or bone artefacts, plant remains are rarely preserved. This has created a paradigm for the Palaeolithic in which plants seldom feature, resulting in a partial and skewed perspective; in fact, plants were as essential to human life then as they are today. Here, we combine morphological and spectroscopic analyses (µ-Raman, µ-FTIR) to provide robust multiscale physical and biomolecular evidence for the deliberate pounding and grinding of Isatis tinctoria L. leaves 34–32,000 years ago. The leaf epidermis fragments were found entrapped in the topography of the used surface of unmodified pebbles, in association with use-wear traces. Although their bitter taste renders them essentially inedible, the leaves have well-recognised medicinal properties and contain indigotin precursors, the chromophore responsible for the blue colour of woad, a plant-based dye that is insoluble in water. We used a stringent approach to contamination control and biomolecular analysis to provide evidence for a new perspective on human behaviour, and the applied technical and ecological knowledge that is likely to have prevailed in the Upper Palaeolithic. Whether this plant was used as a colourant, as medicine, or indeed for both remains unknown, but offers a new perspective on the fascinating possibilities of non-edible plant use.

Background Glucosinolates (GSLs) play important roles in defending against exogenous damage and regulating physiological activities in plants. However, GSL accumulation patterns and molecular regulation mechanisms are largely unknown in Isatis indigotica Fort. Results Ten GSLs were identified in I. indigotica , and the dominant GSLs were epiprogoitrin (EPI) and indole-3-methyl GSL (I3M), followed by progoitrin (PRO) and gluconapin (GNA). The total GSL content was highest (over 20 μmol/g) in reproductive organs, lowest (less than 1.0 μmol/g) in mature organs, and medium in fresh leaves (2.6 μmol/g) and stems (1.5 μmol/g). In the seed germination process, the total GSL content decreased from 27.2 μmol/g (of seeds) to 2.7 μmol/g (on the 120th day) and then increased to 4.0 μmol/g (180th day). However, the content of indole GSL increased rapidly in the first week after germination and fluctuated between 1.13 μmol/g (28th day) and 2.82 μmol/g (150th day). Under the different elicitor treatments, the total GSL content increased significantly, ranging from 2.9-fold (mechanical damage, 3 h) to 10.7-fold (MeJA, 6 h). Moreover, 132 genes were involved in GSL metabolic pathways. Among them, no homologs of AtCYP79F2 and AtMAM3 were identified, leading to a distinctive GSL profile in I. indigotica . Furthermore, most genes involved in the GSL metabolic pathway were derived from tandem duplication, followed by dispersed duplication and segmental duplication. Purifying selection was observed, although some genes underwent relaxed selection. In addition, three tandem-arrayed GSL-OH genes showed different expression patterns, suggesting possible subfunctionalization during evolution. Conclusions Ten different GSLs with their accumulation patterns and 132 genes involved in the GSL metabolic pathway were explored, which laid a foundation for the study of GSL metabolism and regulatory mechanisms in I. indigotica .

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.

Summary A molecular description of lignan biosynthesis in Isatis indigotica displaying its synthetic characteristics and regulatory mechanism is of great importance for the improvement of the production of this class of active compounds. To discover the potential key catalytic steps and regulatory genes, I. indigotica hairy roots elicited by methyl jasmonate (MeJA) were used as a source of systematic variation for exploring the metabolic/transcriptional changes and candidate genes that might play key roles in lignan biosynthesis. The reprogramming modulated by MeJA was classified into three distinct phases, referred to as signal responding, transcriptional activation of metabolic pathways and accumulation of metabolites. Candidate genes were pooled according to the three phases and applied to co‐expression network analysis. In total, 17 genes were identified as hub genes. 4CL3 was selected to validate its impact on lignan biosynthesis. RNAi of 4CL3 resulted in a significant reduction in lignan production. Taken together with its catalytic property, a major route of lignan biosynthesis in I. indigotica was highlighted, which was catalysed by 4CL3 via the esterization of caffeic acid. In conclusion, this study provides new insights into lignan biosynthesis as well as potential targets for metabolic engineering in I. indigotica.

Traditional Chinese medicine relies heavily on herbs, yet there is no information on how these herb plants would respond to climate change. In order to gain insight into such response, we studied the effect of elevated [CO2] on Isatis indigotica Fort, one of the most popular Chinese herb plants. The changes in leaf photosynthesis, chlorophyll fluorescence, leaf ultrastructure and biomass yield in response to elevated [CO2] (550 plus or minus 19 mu mol mol-1) were determined at the Free-Air Carbon dioxide Enrichment (FACE) experimental facility in North China. Photosynthetic ability of I. indigotica was improved under elevated [CO2]. Elevated [CO2] increased net photosynthetic rate (PN), water use efficiency (WUE) and maximum rate of electron transport (Jmax) of upper most fully-expended leaves, but not stomatal conductance (gs), transpiration ratio (Tr) and maximum velocity of carboxylation (Vc,max). Elevated [CO2] significantly increased leaf intrinsic efficiency of PSII (Fv'/Fm') and quantum yield of PSII( Phi PSII), but decreased leaf non-photochemical quenching (NPQ), and did not affect leaf proportion of open PSII reaction centers (qP) and maximum quantum efficiency of PSII (Fv/Fm). The structural chloroplast membrane, grana layer and stroma thylakoid membranes were intact under elevated [CO2], though more starch grains were accumulated within the chloroplasts than that of under ambient [CO2]. While the yield of I. indigotica was higher due to the improved photosynthesis under elevated [CO2], the content of adenosine, one of the functional ingredients in indigowoad root was not affected.

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.