IiAGL6 participates in the regulation of stamen development and pollen formation in Isatis indigotica.

The AGL6 (AGMOUSE LIKE 6) gene is a member of the SEP subfamily and functions as an E-class floral homeotic gene in the development of floral organs. In this study, we cloned IiAGL6, the orthologous gene of AGL6 in Isatis indigotica. The constitutive expression of IiAGL6 in Arabidopsis thaliana resulted in a late-flowering phenotype and the development of curly leaves during the vegetative growth period. Abnormal changes in floral organ development were observed during the reproductive stage. In woad plants, suppression of IiAGL6 using TRV-VIGS (tobacco rattle virus-mediated virus-induced gene silencing) decreased the number of stamens and led to the formation of aberrant anthers. Similar changes in stamen development were also observed in miRNA-AGL6 transgenic Arabidopsis plants. Yeast two-hybrid and BiFC tests showed that IiAGL6 can interact with other MADS-box proteins in woad; thus, playing a key role in defining the identities of floral organs, particularly during stamen formation. These findings might provide novel insights and help investigate the biological roles of MADS transcription factors in I. indigotica.

Genome-wide identification of the NAC transcription factors family and regulation of metabolites under salt stress in Isatis indigotica.

NAC (NAM, ATAF1/2 and CUC2) transcription factors (TFs) are a class of TFs families unique to plants, which not only play an important role in the growth and developmental stages of plants but also function in response to stress and regulation of secondary metabolite biosynthesis. However, there are few studies on NAC genes in the medicinal plant Isatis indigotica. In this study, 96 IiNAC genes were identified based on the whole-genome data of I. indigotica, distributed in seven chromosomes and three contigs. IiNAC genes were structurally conserved and divided into 15 subgroups. Cis-elements were identified in the promoter region of the IiNAC gene in response to plant growth and development, abiotic stresses and hormones. In addition, transcriptome and metabolome data of I. indigotica leaves under salt stress were analyzed to construct a network of IiNAC gene co-expression and metabolite association. Ten differentially expressed IiNAC genes were co-expressed with 109 TFs, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that most of these genes were associated with plant growth and development and abiotic stress responses. Eleven IiNAC genes were positively associated with 72 metabolites. Eleven IiNAC genes were positively or negatively associated with 47 metabolites through 37 TFs. Commonly associated secondary metabolites include two terpenoids, abscisic acid and bilobalide, two flavonoids, dihydrokaempferol and syringaldehyde, a coumarin, 7-methoxycoumarin, an alkaloid, lupinine, and quinone dihydrotanshinone I. This study provides important data to support the identification of the NAC gene family in I. indigotica and the regulatory functions of IiNAC genes in metabolites under salt stress.

IiSVP of Isatis indigotica can reduce the size and repress the development of floral organs.

KEY MESSAGE: IiSVP 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.

Functional identification of the different regions in B-class floral homeotic MADS-box proteins IiAP3 and IiPI from Isatis indigotica.

APETALA3 (AP3) and PISTILLATA (PI) are B-class MADS-box floral homeotic genes of Arabidopsis and are involved in specifying the identity of petals and stamens. In the present work, IiAP3 and IiPI, the respective orthologous genes of AP3 and PI, were cloned from Isatis indigotica. By expressing in ap3-6 and pi-1 homozygous mutant and in wild-type Arabidopsis under the control of AP3 promoter or CaMV 35S promoter, we demonstrated that IiAP3 and IiPI were functionally equivalent to AP3 and PI of Arabidopsis. Referring to previous reports and the research results in the present work, expression patterns of AP3 and PI homologs are not the same in different angiosperms possessing diverse floral structures. It suggests that the alterations in expression may contribute to the changing morphology of flowers. To further determine the relationship between IiAP3 and IiPI, the coding sequences of the different structural regions in these two proteins were swapped with each other, and the data collected from transgenic Arabidopsis plants of the chimeric constructs suggested that MADS domain was irreplaceable for the function of IiAP3, K domain of IiAP3 was involved in specifying the identity of stamens, K domain of IiPI was mainly related to the formation of petals, and C-terminal region of IiPI was involved in characterization of stamens. In addition, a complete KC region of these two proteins was more effective in phenotypic complementation of the mutants.

Isatis tinctoria L. Leaf Extract Inhibits Replicative Senescence in Dermal Fibroblasts by Regulating mTOR-NF-κB-SASP Signaling.

Senescent fibroblasts progressively deteriorate the functional properties of skin tissue. Senescent cells secrete senescence-associated secretory phenotype (SASP) factor, which causes the aging of surrounding non-senescent cells and accelerates aging in the individuals. Recent findings suggested the senomorphic targeting of the SASP regulation as a new generation of effective therapeutics. We investigated whether Isatis tinctoria L. leaf extract (ITE) inhibited senescence biomarkers p53, p21CDKN1A, and p16INK4A gene expression, and SASP secretions by inhibiting cellular senescence in the replicative senescent human dermal fibroblast (RS-HDF). ITE has been demonstrated to inhibit the secretion of SASP factors in several senomorphic types by regulating the MAPK/NF-κB pathway via its inhibitory effect on mTOR. ITE suppressed the inflammatory response by inhibiting mTOR, MAPK, and IκBα phosphorylation, and blocking the nuclear translocation of NF-κB. In addition, we observed that autophagy pathway was related to inhibitory effect of ITE on cellular senescence. From these results, we concluded that ITE can prevent and restore senescence by blocking the activation and secretion of senescence-related factors generated from RS-HDFs through mTOR-NF-κB regulation.

Insights into glucosinolate accumulation and metabolic pathways in Isatis indigotica Fort.

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.

Effects of water and nitrogen coupling on the photosynthetic characteristics, yield, and quality of Isatis indigotica.

Isatis indigotica is a commercial medicinal crop that is widely cultivated with high water and nutrient application, in the arid areas of northwest China. Rational irrigation and nitrogen application are key factors for successful crop management. The objective of this study was to determine the effect of water and nitrogen coupling on the photosynthetic characteristics, yield, and quality of Isatis indigotica produced in northwestern China. Field trials were conducted for 2 consecutive years at an irrigation test station. Data on photosynthetic parameters, yield, and quality were collected from individual Isatis indigotica for each treatment during 2018-2019. The application of nitrogen significantly increased photosynthetic rates and yield under the same irrigation conditions. However, the yields were reduced in the excess water treatments (W3N1 and W3N2) and in the excess nitrogen treatments (W1N3, W2N3, and W3N3) in contrast to the optimum W2N2 treatment. Moreover, the quality indicators of the W2N2 treatment decreased compared with CK, which was due to water stress and more photoassimilates being available to the roots, but the effective quality index value could be effectively improved by greatly increasing the yield.

Structure-based engineering of substrate specificity for pinoresinol-lariciresinol reductases.

Pinoresinol-lariciresinol reductases (PLRs) are enzymes involved in the lignan biosynthesis after the initial dimerization of two monolignols, and this represents the entry point for the synthesis of 8-8' lignans and contributes greatly to their structural diversity. Of particular interest has been the determination of how differing substrate specificities are achieved with these enzymes. Here, we present crystal structures of IiPLR1 from Isatis indigotica and pinoresinol reductases (PrRs) AtPrR1 and AtPrR2 from Arabidopsis thaliana, in the apo, substrate-bound and product-bound states. Each structure contains a head-to-tail homodimer, and the catalytic pocket comprises structural elements from both monomers. ß4 loop covers the top of the pocket, and residue 98 from the loop governs catalytic specificity. The substrate specificities of IiPLR1 and AtPrR2 can be switched via structure-guided mutagenesis. Our study provides insight into the molecular mechanism underlying the substrate specificity of PLRs/PrRs and suggests an efficient strategy for the large-scale commercial production of the pharmaceutically valuable compound lariciresinol.

The effect of NADPH oxidase inhibitor diphenyleneiodonium (DPI) and glutathione (GSH) on Isatis cappadocica, under Arsenic (As) toxicity.

The present work was conducted to assess the effects of arsenic (As, 1000 µM), diphenyleneiodonium (DPI, 10 µM) and reduced glutathione (GSH, 500 µM) on Isatis cappadocica. As treatment decreased plant growth and fresh and dry weight of shoot and root and also enhanced the accumulation of As. As stress also enhanced the oxidative stress biomarkers, hydrogen peroxide (H2O2) and malondialdehyde (MDA) content. However, the application of GSH decreased the content of H2O2 and MDA by 43% and 55%, respectively, as compared to As treatment. The antioxidants like superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX), glutathione reductase (GR) and glutathione S-transferase (GST) also enhanced with As stress. NADPH oxidase inhibitor, the DPI, enhances the effect of As toxicity by increasing the accumulation of As, H2O2, MDA. DPI also enhances the activity of antioxidant enzymes except GR and GST, However, the application GSH increased the plant growth and biomass yield, decreases accumulation of As, H2O2 and MDA content in As as well as As + DPI treated plants. The thiols content [total thiol (TT), non-protein thiol (NPT) protein thiols (PT), and glutathione (GSH)] were decreased in the As + DPI treatment but supplementation of GSH enhanced them. Novelty statement: The study reveals the beneficial role of GSH in mitigating the deleterious effects of Arsenic toxicity through its active involvement in the antioxidant metabolism, thiol synthesis and osmolyte accumulation. Apart from As, We provided the plants NADPH oxidase inhibitor, the diphenyleneiodonium (DPI), which boosts the As toxicity. At present, there is dearth of information pertaining to the effects of DPI on plants growth and their responses under heavy metal stress.GSH application reversed the effect of diphenyleneiodonium (DPI) under As stress preventing the oxidative damage to biomolecules through the modulation of different antioxidant enzymes. The application of GSH for As stressed soil could be a sustainable approach for crop production.

Improved physiological defense responses by application of sodium nitroprusside in Isatis cappadocica Desv. under cadmium stress.

Isatis cappadocica is a well-known arsenic-hyperaccumulator, but there are no reports of its responses to cadmium (Cd). Nitric oxide (NO) is a signaling molecule, which induces cross-stress tolerance and mediates several physio-biochemical processes related to heavy metal toxicity. In this study, the effects of Cd and sodium nitroprusside (SNP as NO donor) on the growth, defense responses and Cd accumulation in I. cappadocica were investigated. When I. cappadocica was treated with 100 and 200 µM Cd, there was an insignificant inhibition of shoot growth. However, Cd stress at Cd400 treatment decreased significantly the dry weight of root and shoot by 73 and 38%, respectively, as compared to control. The application of SNP significantly improved the growth parameters and mitigated Cd toxicity. In addition, SNP decreased reactive oxygen species (ROS) production induced by Cd. The increased total thiol and glutathione (GSH) concentrations after SNP application may play a decisive role in maintaining cellular redox homeostasis, thereby protecting plants against oxidative damage under Cd stress. Bovine hemoglobin (Hb as NO scavenger) reduced the protective role of SNP, suggesting a major role of NO in the defensive effect of SNP. Furthermore, the reduction in shoot growth and the increase of oxidative damage were more severe after the addition of Hb, which confirms the protective role of NO against Cd-induced oxidative stress. The protective role of SNP in decreasing Cd-induced oxidative stress may be related to NO production, which can lead to stimulation of the thiols synthesis and improve defense system.

Hydrogen sulfide decreases Cd translocation from root to shoot through increasing Cd accumulation in cell wall and decreasing Cd2+ influx in Isatis indigotica.

Hydrogen sulfide (H2S), a small gaseous signalling molecule, plays a pivotal role in the plant response to heavy metal stress. Here, we revealed a novel mechanism of Isatis indigotica resistance to cadmium (Cd) stress, in which H2S promotes Cd accumulation in the root and decreases the long-distance transport of Cd from the root to shoot. Cd significantly inhibited Isatis indigotica growth and induced the endogenous H2S level. Application of NaHS (a H2S donor) alleviated the effects of Cd. NaHS restriction of the translocation factor of Cd, elevated the Cd content in roots and depressed the Cd content in shoots. Cd stress decreased the cellulose and pectin contents in the cell wall, but NaHS restored the effect of Cd on the cell wall components. The Cd2+ fluxes were detected by noninvasive microtest technology (NMT). The data showed that NaHS pretreatment decreased the Cd2+ influx and proportion of the Cd content in organelles. We analyzed the effect of NaHS on the metallothionein and phytochelatin (PC) contents in roots and found that the PC and metallothionein1A (MT1A) contents were induced by NaHS. Additionally, the chemical forms of Cd2+ were changed by NaHS. Thus, H2S alters the content of cell wall component, improves Cd accumulation in the cell wall, depresses Cd2+ transmembrane movement, induces the synthesis of metallothioneins and decreases the toxicity of intracellular Cd. Our finding has great value to reduce the loss of Isatis indigotica resulted by heavy metals stress.

Genome-Wide Identification and Analysis on YUCCA Gene Family in Isatis indigotica Fort. and IiYUCCA6-1 Functional Exploration.

Auxin is one of the most critical hormones in plants. YUCCA (Tryptophan aminotransferase of Arabidopsis (TAA)/YUCCA) enzymes catalyze the key rate-limiting step of the tryptophan-dependent auxin biosynthesis pathway, from IPA (Indole-3-pyruvateacid) to IAA (Indole-3-acetic acid). Here, 13 YUCCA family genes were identified from Isatis indigotica, which were divided into four categories, distributing randomly on chromosomes (2n = 14). The typical and conservative motifs, including the flavin adenine dinucleotide (FAD)-binding motif and flavin-containing monooxygenases (FMO)-identifying sequence, existed in the gene structures. IiYUCCA genes were expressed differently in different organs (roots, stems, leaves, buds, flowers, and siliques) and developmental periods (7, 21, 60, and 150 days after germination). Taking IiYUCCA6-1 as an example, the YUCCA genes functions were discussed. The results showed that IiYUCCA6-1 was sensitive to PEG (polyethylene glycol), cold, wounding, and NaCl treatments. The over-expressed tobacco plants exhibited high auxin performances, and some early auxin response genes (NbIAA8, NbIAA16, NbGH3.1, and NbGH3.6) were upregulated with increased IAA content. In the dark, the contents of total chlorophyll and hydrogen peroxide in the transgenic lines were significantly lower than in the control group, with NbSAG12 downregulated and some delayed leaf senescence characteristics, which delayed the senescence process to a certain extent. The findings provide comprehensive insight into the phylogenetic relationships, chromosomal distributions, and expression patterns and functions of the YUCCA gene family in I. indigotica.

Nitric oxide improves tolerance to arsenic stress in Isatis cappadocica desv. Shoots by enhancing antioxidant defenses.

Arsenic (As) is a toxic metalloid that severely hampers plant growth and also poses health risks for humans through the food chain. Although nitric oxide (NO) is known to improve plant resistance to multiple stresses including metal toxicity, little is known about its role in the As tolerance of hyperaccumulator plants. This study investigates the role of the exogenously applied NO donor, sodium nitroprusside (SNP), in improving the As tolerance of Isatis cappadocica, which has been reported to hyperaccumulate As. Exposure to toxic As concentrations significantly increases NO production and damages the cell membrane, as indicated by increased hydrogen peroxide (H2O2) and malondialdehyde (MDA) concentrations, thereby reducing plant growth. However, the addition of SNP improves growth and alleviates As-induced oxidative stress by enhancing the activity of superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR), glutathione S-transferase (GST), glutathione (GSH), as well as proline and thiol concentrations, thereby confirming the beneficial role played by NO in increasing As stress tolerance. Furthermore, the As-induced decrease in growth and the increase in oxidative stress were more marked in the presence of bovine hemoglobin (Hb; a NO scavenger) and N(G)-nitro-l-arginine methyl ester (l-NAME; a NO synthase inhibitor), thus demonstrating the protective role of NO against As toxicity. The reduction in NO concentrations by l-NAME suggests that NOS-like activity is involved in the generation of NO in response to As in I. cappadocica.

Untargeted liquid chromatography coupled with mass spectrometry reveals metabolic changes in nitrogen-deficient Isatis indigotica Fortune.

Isatis indigotica Fortune is a popular herb in traditional Chinese medicine, and various types of metabolites are the basis for its pharmacological efficacy. The biosynthesis and accumulation of these metabolites are closely linked to nitrogen availability; the benefits of low nitrogen application on the environment and herb quality are increasingly prominent. To analyze metabolic changes in the leaves and roots of I.indigotica in nitrogen deficiency conditions, and to identify the pathways and metabolites induced by low nitrogen availability, we used untargeted liquid chromatography coupled with mass spectrometry (UHPLC-TripleTOF) to obtain metabolomics profiling of I.indigotica under two N-deficiency treatments (0 kg/hm2; 337.5 kg/hm2) and normal nitrogen treatment (675 kg/hm2). A total of 447 metabolites were annotated. Principal component analysis separated the three nitrogen treatments. A greater diversity of metabolites was observed in roots than in leaves under N-deficiency treatments, suggesting that roots have a more important function in low N tolerance. Differential metabolites were mainly enriched in purine metabolism, phenylpropanoid biosynthesis, the shikimate pathway, tryptophan metabolism, and flavonoid biosynthesis that notably induced only in leaves in low nitrogen stress. Moderate N-deficiency benefits carbohydrate accumulation, whereas accumulation of most amino acids decreases. Uniquely, L-tryptophan was maintained at a high concentration in N-deficiency conditions. Low nitrogen stress induced the accumulation of some specialized metabolites (matairesinol, dictamnine, 5-hydroxyindoleacetate (serotonin) in roots and vitexin, xanthohumol, sinapyl alcohol in leaves). N-deficiency also increased the accumulation of adenosine and quality indicators of I.indigotica (indirubin-indigo, epigoitrin and anthranilic acid) in a certain degree. Our findings showed that nitrogen deficiency modified roots and leaves conditions of I.indigotica, affecting both the primary and secondary metabolism. Moderate nitrogen reduction was beneficial to the accumulation of active ingredients. Our methods and analysis are expected to provide an insight regarding the diversity of metabolites and regulation of their synthesis in low nitrogen application, and better investigate the nitrogen deficiency effect on I.indigotica.

Metabolic markers for the yield of lipophilic indole alkaloids in dried woad leaves (Isatis tinctoria L.).

The pharmacologically active dichloromethane extracts of dried woad leaves (Isatis tinctoria L.), and the methanol extracts of comparable fresh leaves of the same plants, were analyzed by LC-MSn. The fresh leaf metabolite profile revealed a complex pattern of indolic compounds. Besides the known indigo precursors, isatan A, isatan B and indican, seven previously unreported indole derivatives were characterized: acetylindican, malonylindican, two dioxindole glucosides, dioxindole malonylglucoside, 6-hydroxyindole-3-carboxylic acid 6-O-glucoside and 6-hydroxyindole-3-carboxylic acid glucose ester. The integration of 122 compounds in fresh leaves and of five selected compounds (indoxyl, isatin, indigo, indirubin, and tryptanthrin) in dried leaves, formed the input data for a stepwise modelling procedure generating five predictive linear models. The structure of the predictive models and a cross validation provide evidence that the models could predict well or moderately well the accumulation of the selected lipophilic compounds, and were simple enough to be used in a woad cultivation program. PLS regression models relating each of the five selected dry leaf indolics to the fresh leaf metabolome were then fitted in order to deduct potential precursors and mechanisms leading to the formation of these lipophilic indolics in drying woad leaves. The models suggested glucobrassicin, isatan A and isatan B as the main candidate precursors of these compounds, besides a minor contribution of other fresh leaf indolics, including malonylindican, actylindican and dioxindole malonylglucoside. Dioxindole malonylglucoside was identified here as isatan C. The models further suggested that the accumulation of phenylpropanoid antioxidants in woad leaves has a negative impact on the formation of indoxyl, isatin, indigo, indirubin and tryptanthrin.

Phenylpropanoid profiling reveals a class of hydroxycinnamoyl glucaric acid conjugates in Isatis tinctoria leaves.

The brassicaceous herb, Isatis tinctoria, is an ancient medicinal plant whose rosette leaf extracts have anti-inflammatory and anti-allergic activity. Brassicaceae are known to accumulate a variety of phenylpropanoids in their rosette leaves acting as antioxidants and a UV-B shield, and these compounds often have pharmacological potential. Nevertheless, knowledge about the phenylpropanoid content of I. tinctoria leaves remains limited to the characterization of a number of flavonoids. In this research, we profiled the methanol extracts of I. tinctoria fresh leaf extracts by liquid chromatography - mass spectrometry (LC-MS) and focused on the phenylpropanoid derivatives. We report the structural characterization of 99 compounds including 18 flavonoids, 21 mono- or oligolignols, 2 benzenoids, and a wide spectrum of 58 hydroxycinnamic acid esters. Besides the sinapate esters of malate, glucose and gentiobiose, which are typical of brassicaceous plants, these conjugates comprised a large variety of glucaric acid esters that have not previously been reported in plants. Feeding with 13C6-glucaric acid showed that glucaric acid is an acyl acceptor of an as yet unknown acyltransferase activity in I. tinctoria rosette leaves. The large amount of hydroxycinnamic acid derivatives changes radically our view of the woad metabolite profile and potentially contributes to the pharmacological activity of I. tinctoria leaf extracts.

Chemical Characterization and Biological Activities of Phenolic-Rich Fraction from Cauline Leaves of Isatis tinctoria L. (Brassicaceae) Growing in Sicily, Italy.

The present work focused on the evaluation of the antioxidant and cytotoxic activities of the phenolic-rich fraction (ItJ-EAF) obtained from cauline leaves collected in January from Isatis tinctoria L. (Brassicaceae) growing wild around Acireale (Sicily, Italy). The total phenolic, flavonoid, and condensed tannin contents of the fraction were determined spectrophotometrically, whereas the phenolic profile was assessed by HPLC-PDA/ESI-MS analysis. A total of 20 compounds were positively identified and twelve out of them were never previously reported in I. tinctoria leaves. The fraction exhibited good radical scavenging activity in DPPH test (IC50  = 0.6657 ± 0.0024 mg/ml) and reducing power (3.87 ± 0.71 ASE/ml), whereas, it neither showed chelating activity nor was able to counteract H2 O2 induced oxidative stress damage in Escherichia coli. The antiproliferative effect was evaluated in vitro on two human anaplastic thyroid carcinoma cell lines (CAL-62 and 8505C) by MTT assay. At the highest tested concentration ItJ-EAF significantly reduced (80%) the growth of CAL-62 cells. No cytotoxicity against Artemia salina was observed. It can be concluded that I. tinctoria cauline leaves represent a source of phenolic compounds which could be potentially used as chemopreventive or adjuvant agents against cancer.

Dynamic metabolic and transcriptomic profiling of methyl jasmonate-treated hairy roots reveals synthetic characters and regulators of lignan biosynthesis in Isatis indigotica Fort.

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.

Isatis tinctoria mediated synthesis of amphotericin B-bound silver nanoparticles with enhanced photoinduced antileishmanial activity: A novel green approach.

After malaria, Leishmaniasis is the most prevalent infectious disease in terms of fatality and geographical distribution. The availability of a limited number of antileishmanial agents, emerging resistance to the available drugs, and the high cost of treatment complicate the treatment of leishmaniasis. To overcome these issues, critical research for new therapeutic agents with enhanced antileishmanial potential and low treatment cost is needed. In this contribution, we developed a green protocol to prepare biogenic silver nanoparticles (AgNPs) and amphotericin B-bound biogenic silver nanoparticles (AmB-AgNPs). Phytochemicals from the aqueous extract of Isatis tinctoria were used as reducing and capping agents to prepare silver nanoparticles. Amphotericin B was successfully adsorbed on the surface of biogenic silver nanoparticles. The prepared nanoparticles were characterized by various analytical techniques. UV-Visible spectroscopy was employed to detect the characteristic localized surface plasmon resonance peaks (LSPR) for the prepared nanoparticles. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) studies revealed the formation of spherical silver nanoparticles with an average particle size of 10-20nm. The cubic crystalline structure of the prepared nanoparticles was confirmed by X-ray diffraction (XRD) study. FTIR spectroscopic analysis revealed that plant polyphenolic compounds are mainly involved in metal reduction and capping. Under visible light irradiation, biogenic silver nanoparticles exhibited significant activity against Leishmania tropica with an IC50 value of 4.2µg/mL. The leishmanicidal activity of these nanoparticles was considerably enhanced by conjugation with amphotericin B (IC50=2.43µg/mL). In conclusion, the findings of this study reveal that adsorption of amphotericin B, an antileishmanial drug, to biogenic silver nanoparticles, could be a safe, more effective and economic alternative to the available antileishmanial strategies.

Transcriptome-wide analysis of basic helix-loop-helix transcription factors in Isatis indigotica and their methyl jasmonate responsive expression profiling.

Jasmonates (JAs) act as conserved elicitors of plant secondary metabolism. JAs perception triggers extensive transcriptional reprogramming leading to activation of the entire metabolic pathways. The family of basic helix-loop-helix (bHLH) transcription factors (TFs) has essential roles in JA signaling; however, little is known about their roles in regulation of secondary metabolites in Isatis indigotica. In this study, we identified 78 putative IibHLH sequences using the annotation of I. indigotica transcriptome. The identified proteins were characterized based on phylogenetic and conserved motif analyses. Using RNA sequencing, 16 IibHLHs showed significant positive response to MeJA (methyl jasmonate) at 1h, indicating their roles as early signaling events of JA-mediated transcriptional reprogramming. Ten IibHLHs presented co-expression pattern with biosynthetic pathway genes, suggesting their regulating role in secondary metabolite synthesis. These gene expression profiling data indicate that bHLHs can be used as candidate genes in molecular breeding programs to improve metabolite production in I. indigotica.