Isatis Indigotica Research Articles

IiUGT71B2 catalyzes lignan glycosylation in Isatis indigotica with substrates specificity

Lignans are a major class of secondary metabolites distributed widely in the plant kingdom that are derived from coupling of phenylpropanoid (C6-C3) units. Most lignans are 8–8'-linked, and are often found in their glycosylated forms, which are catalyzed by UDP-dependent glycosyltransferase (UGT). Up to now, all UGTs involved in lignans biosynthesis are determined with substrate promiscuity. Here, IiUGT71B2 is reported from Isatis indigotica that specific catalyzes 4-O and 4′-O glycosylation of furan (lariciresinol) and dibenzylbutyrolactone (matairesinol) type lignans. Transcriptional profile of IiUGT71B2 indicates its involvement in lignan glycosides biosynthesis, which is co-expressed with other lignan biosynthetic genes, and is consistent with metabolic distribution of lignans in organs and tissues as well. Both in vitro and in vivo experiments demonstrate IiUGT71B2 catalyzes the mono- and di- glycosylation of lariciresinol and mono-glycosylation of matairesinol. The ligand docking and site-directed mutagenesis suggest Asn261, Ser289, Ala356, and Tyr395 as key residues, which may determine its catalytic efficiency together with the narrow binding pocket of IiUGT71B2. This study provides insights into lignan metabolism and functional diversity of plant UGTs.

Characterization of isolated starch from Isatis indigotica Fort. root and anhydro-sugars preparation using its decoction residues.

Isatis indigotica Fort. root (Ban-lan-gen, IIR), a traditional Chinese medicine (TCM), has an ancient and well-documented history for its medicinal properties. Aside from epigoitrin, indole alkaloids, and their corresponding derivatives as medicinal ingredients, it also contains lots of biomass such as starch. Herein, a new starch was isolated from IIR and the physicochemical properties such as amylose content, moisture content, ash content, morphology, thermal properties, and crystallography were characterized systematically. The amylose content of IIR starch was 19.84 ± 0.85%, and the size and shape of starch granules is ellipsoidal shape with sizes from 2 to 10μm. IIR starch exhibited a C-type pattern and had 25.92% crystallinity (higher than that of corn starch). The gelatinization temperature of IIR starch was 58.68-75.41°C, and its gelatinization enthalpy was ΔH gel = 4.33J/g. After decocting, the IIR's residues can be used to prepare anhydro-sugars in a polar aprotic solvent. The total carbon yield of levoglucosan (LG), levoglucosenone (LGO), 5-hydroxymethylfurfural (HMF), and furfural (FF) could reach 69.81% from IIR's decoction residues in 1,4-dioxane with 15mM H2SO4 as the catalyst. Further, the residues after dehydration were prepared into biochar by thermochemical conversion and the BET surface area of biochar was 1749.46 m2/g which has good application prospect in soil improvement and alleviates obstacles of IIR continuous cropping.

Insight into tetrahydrofuran lignans from Isatis indigotica fortune with neuroprotective and acetylcholinesterase inhibitor activity

Nine tetrahydrofuran lignans, including three undescribed spiro-lignans, were isolated from Isatis indigotica Fortune (Brassicaceae). Extensive spectroscopic analyses achieved the structure elucidation of these tetrahydrofuran lignans, and quantum chemical calculation combined with the MAEΔΔδ parameter. Notably, isatispironeols A-B have a unique spiro[dienone-tetrahydrofuran] molecular core. These spiro[dienone-tetrahydrofuran] lignans showed comparable neuroprotective effects as the positive control in the H2O2-induced SH-SY5Y cells model. In addition, (−)-(7R,8S,1′R,7′R,8′R)-isatispironeol A possessed more significant AChE inhibitory activity, further interact sites were also predicted by the in silico assay.

Radix Isatidis polysaccharide (RIP) resists the infection of QX-type infectious bronchitis virus via the MDA5/TLR3/IRF7 signaling pathway

Although vaccines play a major role in the prevention of infectious bronchitis (IB), Anti-IB drugs still have great potential in poultry production. Radix Isatidis polysaccharide (RIP) is a crude extract of Banlangen with antioxidant, antibacterial, antiviral, and multiple immunomodulatory functions. The aim of this study was to explore the innate immune mechanisms responsible for RIP-mediated alleviation of infectious bronchitis virus (IBV)-induced kidney lesions in chickens. Specific-pathogen-free (SPF) chicken and chicken embryo kidney (CEK) cells cultures were pretreated with RIP and then infected with the QX-type IBV strain, Sczy3. Morbidity, mortality, and tissue mean lesion scores were calculated for IBV-infected chickens, and the viral loads, inflammatory factor gene mRNA expression levels, and innate immune pathway gene mRNA expression levels in infected chickens and CEK cell cultures were determined. The results show that RIP could alleviate IBV-induced kidney damage, decrease CEK cells susceptibility to IBV infection, and reduce viral loads. Additionally, RIP reduced the mRNA expression levels of the inflammatory factors IL-6, IL-8, and IL-1β by decreasing the mRNA expression level of NF-κB. Conversely, the expression levels of MDA5, TLR3, STING, Myd88, IRF7, and IFN-β were increased, indicating that RIP conferred resistance to QX-type IBV infection via the MDA5, TLR3, IRF7 signaling pathway. These results provide a reference for both further research into the antiviral mechanisms of RIP and the development of preventative and therapeutic drugs for IB.

Woaded Blue: A Colorful Approach to the Dialectic between Written Historical Sources, Experimental Archaeology, Chromatographic Analyses, and Biochemical Research

Research into the sustainability of natural, potentially renewable, resources is one of the major issues of our time. It naturally includes the quest for sustainable sources of colorants for textiles, cosmetics, and food. In industrialized countries, natural dyeing with plants and a few species of coccid insects was practiced on a large scale for centuries before synthetic colorants were developed. Therefore, historical documents on the growing of dye plants and dyeing processes offer a relevant basis from which to start reconsidering the potential of natural colorants in our time. However, written sources need to be completed by experimental archaeologists to allow a scientific understanding of the biochemical reactions at work in the historical processes described. The results of such interdisciplinary research can then inspire contemporary programs to revive the production of natural dyes. The long history of dyeing blue with woad, Isatis tinctoria L., is revisited here as an illustration of the fruitful complementarity of sources and approaches. This article presents a step-by-step re-assessment of the production chain of woad as described in historical texts, from the growing of the plant to its use as a source of indigo in the woad and indigo vats. The experimental reconstitution of the processing of woad leaves into couched woad allowed us to follow the evolution of the composition and proportions of indigoid colorants in the leaves by HPLC analyses. Additionally, HPLC analyses allowed a comparison of the respective indigoid contents of couched woad and sukumo, the form of indigo dye resulting from another couching process, traditionally used in Japan for dyers’ knotweed, Persicaria tinctoria (Ait.) H. Gross. The reconstitution of the 18th century woad and indigo vat process allowed investigations into the bacterial flora associated with the use of couched woad in vat liquors, which were found to contain different indigo-reducing bacteria, including two distinct strains of a new indigo-reducing species.

Physiological and biochemical responses of Isatis indigotica to deficit irrigation in a cold and arid environment.

Water shortage and wastage are critical challenges to sustainable agricultural development, especially in arid and semiarid regions worldwide. Isatis indigotica (woad), as a traditional Chinese herb, was planted in a large area in a cold and arid environment of Hexi. Regulated deficit irrigation can reduce the growth of some vegetative organs by changing the accumulation and distribution of photosynthetic products in crops, thus increasing the economic yield of crops. In agricultural production, crop productivity may be improved by mulched drip irrigation and deficit irrigation. Hence, a field experiment was conducted to investigate the responses of photosynthesis, malondialdehyde, osmotic regulators, antioxidant enzyme activities, and the yield of woad to water deficit at different growth stages. The growth stage of woad was divided in four stages: seedling, vegetative growth, fleshy root growth, and fleshy root maturity. During vegetative growth, fleshy root growth, and fleshy root maturity, three water gradients were set for plants with mild (65-75% in field water capacity, FC), moderate (55-65% in FC), and severe (45-55% in FC) deficits, respectively. In contrast, an adequate water supply (75-85% in FC) during the growth period was designed as the control (CK). The net photosynthetic rate (Pn), transpiration rate, and stomatal conductance of woad significantly decreased (P< 0.05) by moderate and severe water deficits. Still, rehydration after the water deficit could produce a noticeable compensation effect. In contrast, malondialdehyde and proline accumulation significantly increased under moderate and severe water deficits. At the same time, the superoxide dismutase, peroxidase, and catalase all had high activities (increased significantly by 19.87-39.28%, 19.91-34.26%, and 10.63-16.13% compared with CK, respectively), but yields were substantially lower, compared to CK. Additionally, the net photosynthetic rate was negatively correlated with antioxidant enzyme activity. The economic yield of plants subjected to continuous mild water deficit during both vegetative and fleshy root growth was not significantly different from that in CK. Still, the water use efficiency improved significantly. Therefore, the continuous mild water deficit during vegetative and fleshy root growth could improve the physiological and biochemical mechanisms of the plant, representing an optimal irrigation strategy for woad in cold and arid areas.

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

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.

Syringic acid ameliorates testicular oxidative stress via the conservation of endogenous antioxidant markers and inhibition of the activated Nrf2-Keap1-NQO1-HO1 signaling in methyl cellosolve-administered rats

IntroductionSyringic acid (SYRAC) isolated from Ban Lan Gen or Isatis root, a famous Chinese traditional medicine is known to possess antiendotoxic effect. Methyl cellosolve (MCE) on the other hand, is an important constituent of car brake fluids, pesticides, inks, paints, and liquid soaps, rendering humans inevitable to its exposure. MethodsThis study therefore investigated the effect of SYRAC treatments on MCE-induced testicular oxidative stress in rats. Six (6) groups of five (5) rats each were involved in this study that lasted for 30 days. The first group was the control that contained rats served food and water throughout, the second group were administered 100 mg/kg body weight of MCE everyday for 30 days, the third, fourth, and fifth group were treated same way as rats in second group but were treated with 25, 50, and 75 mg/kg body weight of SYRAC respectively for 30 days, while rats in the sixth group were administered 75 mg/kg body weight of SYRAC only. ResultsAt the end of administrations, RTW, testicular GSH level, and activities of GPx, GST, CAT, and SOD were significantly decreased, while MDA, total mRNA expressions of HO-1, NQO1, Keap1, and Nrf2 were significantly increased by MCE compared with control. Compared with MCE only, treatments with SYRAC significantly increased the testicular levels of NO and GSH, as well as activities of GPx, GST, SOD, and CAT, while MDA, mRNA expressions of Keap1, NQO1, Nrf2, and HO-1 were significantly decreased. ConclusionConclusively, MCE-induced testicular oxidative stress was recorded. SYRAC demonstrated a gonadoprotective effect by maintaining the normal levels of the endogenous antioxidants and inhibiting MECE-induced Nrf2 activation and ARE-dependent genes in rats.

Development of Natural Plant Extracts as Sustainable Inhibitors for Efficient Protection of Mild Steel: Experimental and First-Principles Multi-Level Computational Methods.

In the present work, we present the superior corrosion inhibition properties of three plant-based products, Fraxinus excelsior (FEAE), Zingiber zerumbet (ZZAE), and Isatis tinctoria (ITAE), that efficiently inhibit the corrosion of mild steel in phosphoric acid. The anti-corrosion and adsorption characteristics were assessed using a combination of experimental and computational approaches. Weight loss, potentiodynamic polarization, and electrochemical impedance spectroscopy methods were used to evaluate the inhibitive performance of the inhibitors on the metal surface. Then, both DFT/DFTB calculations and molecular dynamic simulations were further adopted to investigate the interaction between organic inhibitor molecules and the metal surface. The protective layers assembled using the active constituents, such as carbonyl and hydroxyl groups, of the three plant-based products offer high electrochemical stability at high temperatures and robust protection against aggressive acidic solutions. All electrochemical measurements showed that the inhibition performance of extracts increased by increasing their concentration and improved in the following order: FEAE &gt; ZZAE &gt; ITAE. Further, these extracts worked as mixed-type inhibitors to block the anodic and cathodic active sites on the mild steel surface. Multi-level computational approaches revealed that FEAE is the most adsorbed inhibitor owing to its ability to provide electron lone pairs for electrophilic reactions. The experimental and theoretical results showed good agreement. These results indicate the possibility of replacing conventional compounds with natural substituted organic products in the fabrication of hybrid materials with effective anti-corrosion performance.

Cloning and functional characterization of lignan glycosyltransferase gene IiUGT349 in Isatis indigotica

Based on the transcriptome data of Isatis indigotica, a total of 110 putative glycosytransferases were identified. Through prokaryotic expression and enzymic activity assay in vitro, a novel lignan glycosyltransferase gene was screened out and named IiUGT349, which catalyzed lariciresinol into lariciresinol-4-O-β-D-glucoside and lariciresinol-4'-O-β-D-glucoside. Bioinformatics analysis suggested that IiUGT349 contained an open reading frame(ORF) of 1 401 bp encoding a protein of 467 amino acids. A protein analysis indicated that IiUGT349 have a predecited molecular weight of 52.77 kDa and pI of 5.96. Phylogenetic analysis showed that IiUGT349 belonging to UGT90 family shared low amino acid sequence identity with the reported lignan glycosyltransferases, which may represent a novel type of lignan glycosyltransferases. Quantitative real-time PCR(qRT-PCR) analysis showed that IiUGT349 was expressed in roots, stems, young leaves and leaves, with the highest expression level in stems. Further biochemical analysis showed that the optimal reaction time of IiUGT349 recombinant protein was 12 h and the optimal temperature was 45 ℃. Subcellular localization demonstrated that IiUGT349 was located in the cytoplasm and nucleus of plants. In this study, a new glucosyltransferase gene IiUGT349 from I. indigotica belonging to the UGT90 family was cloned, which laid a foundation to further investigate its&apos; function and elucidate the lignan glycosides biosynthesis pathway and plays an important role for great significance for the synthetic biology of active lignan glycosides.

Topical Application of Chinese Formula Yeliangen Promotes Wound Healing in Streptozotocin-Induced Diabetic Rats.

Diabetic wound is one of the most severe complications of diabetes mellitus (DM). Despite the associated risks of wound healing impairment in diabetes, treatment strategies remain limited. Yeliangen (YLG) is a Chinese formulation mainly composed of the rhizome of Coptis chinensis, the root of Isatis tinctoria, and the leaf of Isatis indigotica. We investigated the wound healing effects of YLG in type 2 diabetic (T2DM) rats, which were induced by intraperitoneal administration of streptozotocin after a high-fat diet for four weeks. 3 × 3 cm2 full-thickness excisional wounds were created on the dorsal surface of rats and then divided to control (DC), negative (DPJ), positive (DPC), and YLG-treated (DYLG) groups. Rat's wounds were treated twice daily for 21 days. Wound area and wound contraction were detected on days 0, 3, 7, 14, and 21. Histopathological examinations were performed by H&E staining and immunohistochemistry (IHC). The biochemical parameters, mRNAs, and protein expressions were analyzed through enzyme-linked immunoassays (ELISA), qPCR, and western blot, respectively. Compared with other groups, the histological changes of wound tissue in the DYLG group were improved, and the expressions of CD31, eNOS, and PCNA were significantly upregulated. Besides, YLG significantly reduced the inflammatory factors' expressions of TNF-α, NF-κB, MMP-9, and IL-1B on days 7, 14, and 21 postwounding. Moreover, YLG induced angiogenesis and neovascularization by significantly increasing the levels of VEGF, TGF-β1, EGF, PDGF, and SDF-1α on days 3, 7, and 14. In conclusion, YLG improved wound healing by reducing inflammation and increasing angiogenesis which may provide an alternative and effective approach for diabetic wound therapy.

Preparation of Hydrogels Based Radix Isatidis Residue Grafted with Acrylic Acid and Acrylamide for the Removal of Heavy Metals

A series of hydrogels as biosorbents to remove heavy metal ions (Pb2+, Cu2+, and Cd2+) were prepared using Radix Isatidis residues as material grafted with acrylic acid and acrylamide. The surfaces of Radix Isatidis residue/acrylic acid-co-acrylamide (RIR/AA-co-AM), Radix Isatidis residue/polyacrylamide (RIR/PAM3), and Radix Isatidis residue/polyacrylic acid (RIR/PAA4) hydrogels have a sponge-like, three-dimensional, and highly microporous structure. The hydrogels all have considerable swelling properties and the swelling rate of RIR/PAA4 is the highest at 9240%. The hydrogels all possess high adsorptivity to Pb2+, Cu2+, and Cd2+. Under optimized conditions, the maximum adsorption capacity of RIR/AA-co-AM hydrogel is 655.4 mg/g for Pb2+, 367.2 mg/g for Cd2+, and 290.5 mg/g for Cu2+. The maximum adsorption capacity of RIR/AA-co-AM hydrogel for Cd2+ and Cu2+ is slightly lower than that of RIR/PAA4. In addition, the adsorption process of RIR/AA-co-AM for heavy metal ions conforms with the pseudo-second-order kinetic equation and Langmuir adsorption isotherm. Based on the microstructure analysis and adsorption kinetics, electrostatic adsorption and ion exchange are identified as the mechanisms for the hydrogels removal of heavy metal ions from water. It infers that hydrogels from Chinese herb residue can be used to effectively remove heavy metals from wastewater and improve the reutilization of Chinese herb residue.

Identification and investigation of a novel NADP+-dependent secoisolariciresinol dehydrogenase from Isatis indigotica.

Cofactors are crucial for the biosynthesis of natural compounds, and cofactor engineering is a useful strategy for enzyme optimization due to its potential to enhance enzyme efficiency. Secoisolariciresinol dehydrogenase (SIRD) was reported to convert secoisolariciresinol into matairesinol in an NAD+-dependent reaction. Here, a SIRD designated as IiSIRD2 identified from Isatis indigotica was found to utilize NADP+ as the cofactor. To explore the structural basis for this unique cofactor preference, model-based structural analysis was carried out, and it was postulated that a variation at the GXGGXG glycine-rich motif of IiSIRD2 alters its cofactor preference. This study paves way for future investigations on SIRD cofactor specificity and cofactor engineering to improve SIRD's catalytic efficiency.

A focused natural compound screen reveals senolytic and senostatic effects of Isatis tinctoria

ABSTRACT Natural products and their derivatives historically represent alternatives to conventional synthetic molecules for pharmacotherapy, ranging from cancer chemotherapeutics to cosmetic ingredients that exert anti-aging activities. Cellular senescence is considered a main driver of skin aging, yet natural products that target skin senescence in a specific manner are not thoroughly explored. Here, we performed a focused compound screen to identify natural products that exert anti-senescence effects. We found that Isatis tinctoria, woad extracts, displayed a senolytic effect on senescent human skin fibroblasts. Furthermore, treatment with woad extracts attenuated the expression of pro-inflammatory senescence-associated secretory phenotype (SASP), showing a senostatic activity. Intriguingly, woad extracts displayed only a marginal cytotoxic effect toward senescent human lung fibroblasts. Thus, our results reveal the potential activities of woad extracts for targeting skin senescence and suggest that woad extracts could be an attractive ingredient for cosmetics to prevent skin aging.

A comprehensive comparison on root yield and content of polysaccharides and alkaloids from ten germplasms of Isatis indigotica Fort. in two growing areas

In order to promote the application of high-quality varieties and explore the diversity of cultivated resources of Isatis indigotica Fort., the source of medicinal Banlangen, 10 germplasms were planted in Heilongjiang (HLJ) and Shanxi (SX) provinces to analyze two-year agronomic traits, yields and active ingredient contents in this study. The results showed that germplasm no. 2 had the highest yields in both HLJ (236.54 kg/mu) and SX (368.68 kg/mu), and it had the highest Epigoitrin (RSG) content in HLJ in 2020 (0.8946 %). In addition, the polysaccharide (IRPS) content of germplasm no. 2 was the highest in HLJ (up to 39.100 mg/g), and ranked second in SX (30.767 mg/g). It was the only germplasm with IRPS content that exceeded 30 mg/g in both areas. The yield and RSG content of germplasm no. 6 was slightly lower than that of germplasm no. 2. The germplasms no. 2 and 6 had remarkable advantages in yield and active component content which were worthy of promotion as excellent cultivars in planting industry. Germplasm no. 5 and 10 had advantages in root width and IRPS content, respectively, which could be used as breeding germplasms. Screening germplasm with excellent traits and promoting its cultivation will help to ensure the quality stability of medicinal materials, and to improve economic efficiency of Banlangen production.

Deficit irrigation decision-making of indigowoad root based on a model coupling fuzzy theory and grey relational analysis

Water scarcity is a critical constraint to the cultivation of medicinal crops. Establishing irrigation requirements for medicinal plants is crucial for the large-scale production of crude drugs in China. To evaluate the response of indigowoad root growth to water deficit (WD) under-mulched drip irrigation, and determine the optimal deficit irrigation mode for this medicinal crop in Hexi Corridor’s desert oasis, two WD levels were applied: mild (65–75% FC [field capacity]) and moderate (55–65% FC). These treatments were implemented during the vegetative growth (VG) and fleshy-root growth (RG) stages, and sufficient water supply maintained during the other plant stages. A total of seven combined treatments were imposed, with sufficient water supply (85–75% FC) as the control treatment. From the 2-year-long (2017, 2018) field experiment’s results, 12 indicators of indigowoad root growth, yield, water consumption and quality attributes were evaluated via weighted fuzzy evaluation and a grey relation-coupling model. Results indicated that, with greater irrigation amounts throughout the plant growth period, the fuzzy evaluation value of growth, yield and water consumption indicators increased whereas their quality indicators decreased. The fuzzy evaluation value of yield indicators of V1G0 (mild WD treatment during VG) was the highest (0.1582, 0.1581), while that of growth indicators was also relatively high (0.1524, 0.1532). Fuzzy evaluation values of growth and yield indicators of V1G1 (mild WD during VG and RG) exceeded those of V1G2 (mild WD during VG and moderate WD during RG), but evaluation value of quality indicators was lower for V1G1 (0.1457, 0.1457) than V1G2 (0.1494, 15155). Based on the weighted grey relation model, the comprehensive score of V1G0 was deemed best. Therefore, to ensure both yield and quality, the V1G1 is recommended. Our results can provide a scientific basis for efficient water-saving cultivation of indigowoad root in Hexi oasis regions.

Identification of the bZIP gene family and regulation of metabolites under salt stress in isatis indigotica.

The bZIP transcription factor family plays important roles in plant growth and development, response to stress, and regulation of secondary metabolite biosynthesis. The identification and molecular function of bZIP gene have been deeply studied in the model plant Arabidopsis thaliana, but it has not been reported in the medicinal plant Isatis indigotica. In this study, 65 IibZIP genes were identified in the genome of I. indigotica, which were distributed on seven chromosomes, were highly conserved, could be classified into 11 subgroups. Transcriptomic and metabolomic data for leaves of I. indigotica exposed to salt stress were analyzed to construct an IibZIP gene co-expression network and metabolite correlation network. Seventeen IibZIP genes were co-expressed with 79 transcription factors, and GO and KEGG enrichment analysis showed that most of these genes were associated with abiotic stress and hormone responses of plants. 17 IibZIP genes regulated 110 metabolites through 92 transcription factor associations. In addition, IibZIP23, IibZIP38 and IibZIP51 were associated with six metabolites including three alkaloids (quinoline alkaloid stylopine, indole alkaloids tabersonine and indole-3-acetic acid), flavonoid myricetin 3-O-galactoside, and two primary metabolites 2-hydroxy-6-aminopurine, 3-dehydroshikimic acid were strongly correlated. This study provides data for identification of the IibZIP gene family and their regulation of metabolites in response to salt stress.

Deficit mulched drip irrigation improved yield and quality while reduced water consumption of isatis indigotica in a cold and arid environment.

Deficit irrigation is an effective alternative to traditional irrigation, as it improves crop productivity and conserves water. However, crops may be sensitive to deficit irrigation-induced water stress at different periods. To access the effect of deficit irrigation on the growth, water consumption characteristics, yield, and quality of Isatis indigotica (woad), we performed a three-year (2017-2019) mulched drip irrigation field experiment. Woad plants were provided adequate water supply at the seedling stage but were subjected to mild (65–75% field water capacity FC), moderate (55–65% FC), and severe (45–55% FC) water deficit at the vegetative growth, fleshy root growth and fleshy root maturity stages, respectively; plants supplied with adequate water throughout the growth period served as a control (CK, 75–85% FC). The water consumption characteristics, agronomic traits, dry matter content and distribution, yield, and quality of these plants were measured at various growth stages. The results showed that the total water consumption in water deficit was significantly less than that in CK by 4.44–10.21% (P< 0.05). The dry matter content of plants treated with moderate (WT2 and WT5) and severe (WT3) water deficit was reduced by 12.83–28.75%. The economic yield of mild water deficit-treated plants was higher during vegetative growth (WT1) and fleshy root growth (WT4), while the water use efficiency of these plants was significantly increased by 7.84% and 6.92% at the two growth stages, respectively. Continuous mild water deficit (WT4) enhanced the contents of indigo, indirubin, (R,S)-goitrin, polysaccharides, and soluble proteins during vegetative growth and fleshy root growth, while moderate and severe water deficit were detrimental to the quality of woad plants. Thus, continuous mild water deficit during vegetative and fleshy root growth periods (WT4) is optimal for the cultivation of woad in the cold and cool irrigation district of the Hexi Oasis region.

Genome-wide analysis of AP2/ERF superfamily in Isatis indigotica

ObjectiveAP2/ERF (APETALA2/ethylene-responsive factor) superfamily is one of the largest gene families in plants and has been reported to participate in various biological processes, such as the regulation of biosynthesis of active lignan. However, few studies have investigated the genome-wide role of the AP2/ERF superfamily in Isatis indigotica. This study establishes a complete picture of the AP2/ERF superfamily in I. indigotica and contributes valuable information for further functional characterization of IiAP2/ERF genes and supports further metabolic engineering. MethodsTo identify the IiAP2/ERF superfamily genes, the AP2/ERF sequences from Arabidopsis thaliana and Brassica rapa were used as query sequences in the basic local alignment search tool. Bioinformatic analyses were conducted to investigate the protein structure, motif composition, chromosome location, phylogenetic relationship, and interaction network of the IiAP2/ERF superfamily genes. The accuracy of omics data was verified by quantitative polymerase chain reaction and heatmap analyses. ResultsOne hundred and twenty-six putative IiAP2/ERF genes in total were identified from the I. indigotica genome database in this study. By sequence alignment and phylogenetic analysis, the IiAP2/ERF genes were classified into 5 groups including AP2, ERF, DREB (dehydration-responsive element-binding factor), Soloist and RAV (related to abscisic acid insensitive 3/viviparous 1) subfamilies. Among which, 122 members were unevenly distributed across seven chromosomes. Sequence alignment showed that I. indigotica and A. thaliana had 30 pairs of orthologous genes, and we constructed their interaction network. The comprehensive analysis of gene expression pattern in different tissues suggested that these genes may play a significant role in organ growth and development of I. indigotica. Members that may regulate lignan biosynthesis in roots were also preliminarily identified. Ribonucleic acid sequencing analysis revealed that the expression of 76 IiAP2/ERF genes were up- or down-regulated under salt or drought treatment, among which, 33 IiAP2/ERF genes were regulated by both stresses. ConclusionThis study undertook a genome-wide characterization of the AP2/ERF superfamily in I. indigotica, providing valuable information for further functional characterization of IiAP2/ERF genes and discovery of genetic targets for metabolic engineering.

Multi-analytical study of the medieval wall paintings from the rupestrian church Grotta del Crocifisso at Lentini (eastern Sicily): new evidence of the use of woad (Isatis tinctoria)

This study presents the results of the examination and characterisation of the wall paintings that decorate the rupestrian church named Grotta del Crocifisso, which is located in the territory of Lentini (eastern Sicily, few tens of kilometres from Catania and Syracuse). The earliest mural paintings in the church date back to the twelfth century AD. A multi-analytical approach was adopted for the characterisation of stone materials, secondary degradation products, and pigments. For this purpose, the following techniques were used: reflected light microscopy (RLM), polarised light microscopy on thin sections (PLM), X-ray powder diffraction (XRPD), mercury intrusion porosimetry (MIP), portable X-ray fluorescence (p-XRF), scanning electron microscopy (SEM-EDS), Fourier-transform infrared spectroscopy (FTIR), and Raman spectroscopy (RS). The lithic substrate and the plaster’s coating layers were thoroughly characterised from compositional and textural points of view, and the use of locally available raw materials was established. Similarly, the newly formed crystalline phases produced by alteration processes of the original materials were recognised. The red, yellow, brown, and green pigments were easily identified by p-XRF and SEM-EDS. The use of “earth pigments” widely available in the surrounding area (various types of ochre) was thus highlighted. The recognition of the dark blue pigment created some additional issues in its identification, making further diagnostic methods necessary. In fact, the use of the most common mineral pigments was categorically excluded by both p-XRF and SEM-EDS, since no chromophore metallic elements were highlighted with the exception of trace amounts of iron. A combination of detailed microscopic observations together with the application of FTIR and RS supported the use of an organic pigment obtained from the maceration of woad (Isatis tinctoria). The green pigment is the result of a mixture between woad and yellow ochre. Woad is even today easily available in Sicily, and some additional experimental tests were carried out on Isatis tinctoria that had been freshly collected in the area (treated with traditional procedures). Over the past centuries, woad was widely used for dyeing fabrics, but its practice for wall paintings has only been sporadically proven. The identification reported in this case study could be considered a novelty at least in the Sicilian panorama.