[Identification and expression analysis of whole gene family of Isatis indigotica 4-coumarate: CoA ligase].

The 4-coumarate: CoA ligase(4CL) is a key enzyme in the upstream pathway of phenylpropanoids such as flavonoids, soluble phenolic esters, lignans, and lignins in plants. In this study, 13 4CL family members of Arabidopsis thaliana were used as reference sequences to identify the 4CL gene family candidate members of Isatis indigotica from the reported I. indigotica genome. Further bioinformatics analysis and analysis of the expression pattern of 4CL genes and the accumulation pattern of flavonoids were carried out. Thirteen 4CL genes were obtained, named Ii4CL1-Ii4CL13, which were distributed on chromosomes 1, 2, 3, 4, and 6. The analysis of the gene structure and conserved structural domains revealed the intron number of I. indigotica 4CL genes was between 1 and 12 and the protein structural domains were highly conserved. Cis-acting element analysis showed that there were multiple response elements in the promoter sequence of I. indigotica 4CL gene family, and jasmonic acid had the largest number of reaction elements. The collinearity analysis showed that there was a close relationship between the 4CL gene family members of I. indigotica and A. thaliana. As revealed by qPCR results, the expression analysis of the 4CL gene family showed that 10 4CL genes had higher expression levels in the aboveground part of I. indigotica. The content assay of flavonoids in different parts of I. indigotica showed that flavonoids were mainly accumulated in the aboveground part of plants. This study provides a basis for further investigating the roles of the 4CL gene family involved in the biosynthesis of flavonoids in I. indigotica.

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.

The potential mechanism of response to light intensity in energy metabolism mediated by miRNA in Isatis indigotica.

Light is the main source of energy for plant growth. Studies have shown that I. indigotica is a light-demanding plant and its yield and various active components are positively correlated with light intensity, but no studies of light intensity affecting energy metabolism in I. indigotica have been reported. Mitochondria are the main site of energy metabolism, and miRNAs are important factors in regulating gene expression, this experiment attempts to study the effects of different light intensities on energy metabolism from the perspective of mitochondria and miRNAs. The results show that the biomass、mitochondrial structural integrity and energy metabolism in I. indigotica were found to be positively correlated with light intensity. Small RNA and transcriptome sequencing identified 241 miRNAs and 36,372 mRNAs, and degradomic technology identified 72 miRNAs targeting 106 mRNAs, among which 12 pairs of miRNA-mRNAs were annotated on mitochondria. Combined with RT-qPCR validation, it was concluded that miR167a-5p positively regulates LETM1 and affects mitochondrial structure, miR400-5p and mIR169m-p3_1ss15CT negatively regulate GRXS15 and CMC4, respectively, affecting SDH and CCO activities, and miR395a-APS4 may affect the utilization of ATP and sulfate assimilation. In summary, the results of this study complement and enrich knowledge of light effects on mitochondria from the perspective of miRNA, while providing guidance for the cultivation of I. indigotica.

Silver and salicylic acid-chitosan nanoparticles alter indole alkaloid production and gene expression in root and shoot cultures of Isatis tinctoria and Isatis ermenekensis.

Isatis spp. are well-known for their industrial significance due to natural sources of indigotin and indirubin, important indole alkaloids, used in the dye and pharmaceutical industries. In this study, silver nanoparticles (AgNP) and salicylic acid-chitosan nanoparticles (SA-CNP) were synthesized and applied to enhance the production of indigotin and indirubin in shoot and root cultures of Isatis tinctoria and Isatis ermenekensis. Different doses of AgNP and SA-CNP were administered to three-week-old shoot and root cultures, and the effects were assessed at 12, 24, and 48 h. The harvested samples were analyzed to quantify indigotin and indirubin levels. Furthermore, the expression levels of It-TSA and CYP79B2 genes, known to be involved in indole alkaloid biosynthesis, were determined. In I. tinctoria roots, the highest levels of indigotin and indirubin were observed after applying 150 mg L-1 of SA-CNP for 48 h while in I. ermenekensis shoots, indigotin and indirubin reached the maximum levels with the application of 8 mg L-1 AgNP for 48 h. NP application had no remarkable effects on the accumulation of indigotin and indirubin in I. tinctoria shoots and I. ermenekensis roots compared to controls. Additionally, shoot cultures demonstrated superior indirubin production, which significantly increased with AgNP applications. The gene expression analysis also exhibited significant correlations with the changes in indigotin and indirubin levels. The findings of this study lay the groundwork for enhancing in vitro production of indigotin and indirubin in Isatis species through NP applications, and for developing high-capacity production strategies by determining optimal dosages in scale-up studies.

Classification of Isatis indigotica Fortune and Isatis tinctoria Linnaeus via comparative analysis of chloroplast genomes.

BACKGROUND: Isatis tinctoria Linnaeus and Isatis indigotica Fortune are very inconsistent in their morphological characteristics, but the Flora of China treats them as the same species. In this work, a new technology that differs from conventional barcodes is developed to prove that they are different species and to clarify their classification. RESULTS AND METHODS: I. indigotica was indistinguishable from I. tinctoria when using ITS2. CPGAVAS2 was used to construct the chloroplast genomes. MAFFT and DnaSP were used to calculate nucleotide polymorphism, the chloroplast genomes of the two have high diversity in the rpl32 ~ trnL-UAG short region. When using this region as a mini barcode, it was found that there are obvious differences in the base numbers of I. tinctoria and different ploidy I. indigotica were found, but diploid and tetraploid I. indigotica had the same number of bases. Moreover, the reconstruction of the maximum likelihood (ML) tree, utilizing the mini-barcode, demonstrated that I. tinctoria and both diploid and tetraploid I. indigotica are located on distinct branches. The genome size of tetraploid I. indigotica was approximately 643.773 MB, the heterozygosity rate was approximately 0.98%, and the repeat sequence content was approximately 90.43%. This species has a highly heterozygous, extremely repetitive genome. CONCLUSION: A new method was established to differentiate between I. indigotica and I. tinctoria. Furthermore, this approach provides a reference and basis for the directional breeding of Isatis.

[Cloning and catalytic analysis of Isatis indigotica chalcone isomerase in vitro].

Chalcone isomerase is a key rate-limiting enzyme in the biosynthesis of flavonoids in higher plants, which determines the production of flavonoids in plants. In this study, RNA was extracted from different parts of Isatis indigotica and reverse-transcribed into cDNA. Specific primers with enzyme restriction sites were designed, and a chalcone isomerase gene was cloned from I. indigotica, named IiCHI. IiCHI was 756 bp in length, containing a complete open reading frame and encoding 251 amino acids. Homology analysis showed that IiCHI was closely related to CHI protein of Arabidopsis thaliana and had typical active sites of chalcone isomerase. Phylogenetic tree analysis showed that IiCHI was classified into type Ⅰ CHI clade. Recombinant prokaryotic expression vector pET28a-IiCHI was constructed and purified to obtain IiCHI recombinant protein. In vitro enzymatic analysis showed that the IiCHI protein could convert naringenin chalcone into naringenin, but could not catalyze the production of liquiritigenin by isoliquiritigenin. The results of real-time quantitative polymerase chain reaction(qPCR) showed that the expression level of IiCHI in the aboveground parts was higher than that in the underground parts and the expression level was the highest in the flowers of the aboveground parts, followed by leaves and stems, and no expression was observed in the roots and rhizomes of the underground parts. This study has confirmed the function of chalcone isomerase in I. indigotica and provided references for the biosynthesis of flavonoid components.

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.

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

OBJECTIVE: AP2/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. METHODS: To 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. RESULTS: One 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. CONCLUSION: This 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.

Cloning and catalytic analysis of Isatis indigotica chalcone isomerase in vitro / 中国中药杂志

Chalcone isomerase is a key rate-limiting enzyme in the biosynthesis of flavonoids in higher plants, which determines the production of flavonoids in plants. In this study, RNA was extracted from different parts of Isatis indigotica and reverse-transcribed into cDNA. Specific primers with enzyme restriction sites were designed, and a chalcone isomerase gene was cloned from I. indigotica, named IiCHI. IiCHI was 756 bp in length, containing a complete open reading frame and encoding 251 amino acids. Homology analysis showed that IiCHI was closely related to CHI protein of Arabidopsis thaliana and had typical active sites of chalcone isomerase. Phylogenetic tree analysis showed that IiCHI was classified into type Ⅰ CHI clade. Recombinant prokaryotic expression vector pET28a-IiCHI was constructed and purified to obtain IiCHI recombinant protein. In vitro enzymatic analysis showed that the IiCHI protein could convert naringenin chalcone into naringenin, but could not catalyze the production of liquiritigenin by isoliquiritigenin. The results of real-time quantitative polymerase chain reaction(qPCR) showed that the expression level of IiCHI in the aboveground parts was higher than that in the underground parts and the expression level was the highest in the flowers of the aboveground parts, followed by leaves and stems, and no expression was observed in the roots and rhizomes of the underground parts. This study has confirmed the function of chalcone isomerase in I. indigotica and provided references for the biosynthesis of flavonoid components.

Genome-wide analysis of AP2/ERF superfamily in Isatis indigotica / 中西医结合学报

OBJECTIVE@#AP2/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.@*METHODS@#To 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.@*RESULTS@#One 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.@*CONCLUSION@#This 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.

(-)-Lariciresinol Isolated from the Roots of Isatis indigotica Fortune ex Lindl. Inhibits Hepatitis B Virus by Regulating Viral Transcription.

Chronic hepatitis induced by hepatitis B virus (HBV) infection is a serious public health problem, leading to hepatic cirrhosis and liver cancer. Although the currently approved medications can reliably decrease the virus load and prevent the development of hepatic diseases, they fail to induce durable off-drug control of HBV replication in the majority of patients. The roots of Isatis indigotica Fortune ex Lindl., a traditional Chinese medicine, were frequently used for the prevention of viral disease in China. In the present study, (-)-lariciresinol ((-)-LRSL), isolated from the roots of Isatis indigotica Fortune ex Lindl., was found to inhibit HBV DNA replication of both wild-type and nucleos(t)ide analogues (NUCs)-resistant strains in vitro. Mechanism studies revealed that (-)-LRSL could block RNA production after treatment, followed by viral proteins, and then viral particles and DNA. Promoter reporter assays and RNA decaying dynamic experiments indicated that (-)-LRSL mediated HBV RNA reduction was mainly due to transcriptional inhibition rather than degradation. Moreover, (-)-LRSL in a dose-dependent manner also inhibited other animal hepadnaviruses, including woodchuck hepatitis virus (WHV) and duck hepatitis B virus (DHBV). Combining the analysis of RNA-seq, we further found that the decrease in HBV transcriptional activity by (-)-LRSL may be related to hepatocyte nuclear factor 1α (HNF1α). Taken together, (-)-LRSL represents a novel chemical entity that inhibits HBV replication by regulating HNF1α mediated HBV transcription, which may provide a new perspective for HBV therapeutics.

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.

SEPALLATA--like genes of Isatis indigotica can affect the architecture of the inflorescences and the development of the floral organs.

BACKGROUND: The architecture of inflorescence and the development of floral organs can influence the yield of seeds and have a significant impact on plant propagation. E-class floral homeotic MADS-box genes exhibit important roles in regulation of floral transition and differentiation of floral organs. Woad (Isatis indigotica) possesses unique inflorescence, floral organs and fruit. However, very little research has been carried out to determine the function of MADS-box genes in this medicinal cruciferous plant species. RESULTS: SEPALLATA orthologs in I. indigotica were cloned by degenerate PCR. The sequence possessing the highest identity with SEP2 and SEP4 of Arabidopsis were named as IiSEP2 and IiSEP4, respectively. Constitutive expression of IiSEP2 in Columbia (Col-0) ecotype of Arabidopsis led to early flowering, and the number of the flowers and the lateral branches was reduced, indicating an alteration in architecture of the inflorescences. Moreover, the number of the floral organs was declined, the sepals were turned into carpelloid tissues bearing stigmatic papillae and ovules, and secondary flower could be produced in apetalous terminal flowers. In 35S::IiSEP4-GFP transgenic Arabidopsis plants in Landsberg erecta (Ler) genetic background, the number of the floral organs was decreased, sepals were converted into curly carpelloid structures, accompanied by generation of ovules. Simultaneously, the size of petals, stamens and siliques was diminished. In 35S::IiSEP4-GFP transgenic plants of apetalous ap1 cal double mutant in Ler genetic background, the cauliflower phenotype was attenuated significantly, and the petal formation could be rescued. Occasionally, chimeric organs composed of petaloid and sepaloid tissues, or petaloid and stamineous tissues, were produced in IiSEP4 transgenic plants of apl cal double mutant. It suggested that overexpression of IiSEP4 could restore the capacity in petal differentiation. Silencing of IiSEP4 by Virus-Induced Gene Silencing (VIGS) can delay the flowering time, and reduce the number and size of the floral organs in woad flowers. CONCLUSION: All the results showed that SEPALLATA-like genes could influence the architecture of the inflorescence and the determinacy of the floral meristems, and was also related to development of the floral organs.

[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-ß-D-glucoside and lariciresinol-4'-O-ß-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' function and elucidate the lignan glycosides biosynthesis pathway and plays an important role for great significance for the synthetic biology of active lignan glycosides.

Integrative analyses of targeted metabolome and transcriptome of Isatidis Radix autotetraploids highlighted key polyploidization-responsive regulators.

BACKGROUND: Isatidis Radix, the root of Isatis indigotica Fort. (Chinese woad) can produce a variety of efficacious compound with medicinal properties. The tetraploid I. indigotica plants exhibit superior phenotypic traits, such as greater yield, higher bioactive compounds accumulation and enhanced stress tolerance. In this study, a comparative transcriptomic and metabolomic study on Isatidis Radix autotetraploid and its progenitor was performed. RESULTS: Through the targeted metabolic profiling, 283 metabolites were identified in Isatidis Radix, and 70 polyploidization-altered metabolites were obtained. Moreover, the production of lignans was significantly increased post polyploidization, which implied that polyploidization-modulated changes in lignan biosynthesis. Regarding the transcriptomic shift, 2065 differentially expressed genes (DEGs) were identified as being polyploidy-responsive genes, and the polyploidization-altered DEGs were enriched in phenylpropanoid biosynthesis and plant hormone signal transduction. The further integrative analysis of polyploidy-responsive metabolome and transcriptome showed that 1584 DEGs were highly correlated with the 70 polyploidization-altered metabolites, and the transcriptional factors TFs-lignans network highlighted 10 polyploidy-altered TFs and 17 fluctuated phenylpropanoid pathway compounds. CONCLUSIONS: These results collectively indicated that polyploidization contributed to the high content of active compounds in autotetraploid roots, and the gene-lignan pathway network analysis highlighted polyploidy-responsive key functional genes and regulators.

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.

Identification and expression analysis of the WRKY gene family in Isatis indigotica.

The WRKY proteins, which represent one of the largest families of transcriptional regulators in plants, play pivotal roles in regulating multiple processes of growth and development, particularly in diverse stress responses. Isatis indigotica is widely used in Traditional Chinese Medicine and is famous for its use as a dye for the color indigo. However, reports of the WRKY gene family in I. indigotica are limited. In this study, 64 IiWRKY genes encoding proteins with the complete WRKY domain were identified from genome of I. indigotica. Based on their structure and phylogenetic relationships of this gene family in I. indigotica, the IiWRKY genes were classified into three groups: Group I (n = 13), Group II (n = 35) and Group III (n = 16). Sequence alignment revealed that IiWRKY proteins harbored two variants, WRKYRQK and WRKYGKK, of the highly conserved WRKYGQK motif. The number of exons in IiWRKY genes varied from two to 14, with most of IiWRKY genes containing three exons. Investigation of gene duplication demonstrated that 10 and 14 IiWRKY genes were incorporated in tandem and segmental duplication events, respectively. Finally, the expression profiles derived from transcriptome data and quantitative real-time PCR analysis showed distinct expression patterns of these IiWRKY gene in five different organs or in response to four abiotic stresses. Taken together, our results will contribute to functional analysis of IiWRKY genes, and also provide a basis for further clarification of the molecular mechanism of stress responses in this important herb.

Development of the species-specific multiplex PCR and DNA sequencing methods for rapid authentication of Isatidis Folium and its adulterants.

Isatis indigotica Fort. (family Cruciferae), is an herb widely used in traditional herbal medicine and its dried leave was named "ISATIDIS FOLIUM". Baphicacanthus cusia (Ness) Bremek. and Polygonum tinctorium Ait. are commonly misused as ISATIDIS FOLIUM in Chinese Medicine pharmacy. For the purpose of being not misused, specific primers based on the sequence difference of chloroplast trnH-psbA intergenic spacer were designed and multiplex polymerase chain reaction method (multiplex PCR) was developed. In this study, 29 original herbal materials were analyzed and our results show that DNA size after multiplex PCR was able to distinguish variations between three herbs. DNA fragments of 464, 297, 170 base pairs (bps) were represented for I. indigotica and B. cusia and P. tinctorium, respectively. In conclusion, our investigations demonstrate that molecular identification method provides more accurate results for medicinal plants detection and good quality control of ISATIDIS FOLIUM.

Cloning of a SEPALLATA4-like gene (IiSEP4) in Isatis indigotica Fortune and characterization of its function in Arabidopsis thaliana.

E-class MADS-box genes, SEPALLATA (SEP), participate in various aspects of plant development together with B-, C- and D-class MADS-box genes. IiSEP4, a homologous gene of SEP4, was cloned from Isatis indigotica. IiSEP4 was highly expressed in sepals, and its mRNA was mildly detected in leaves, inflorescences, flowers, stamens and young silicles. Constitutive expression of IiSEP4 in Arabidopsis thaliana caused early flowering, accompanied by the reduction of flowers and floral organs. Moreover, the sepals in some flowers were transformed into carpelloid structures with stigmatic papillae, and obviously accompanied by ovule formation. Yeast two-hybrid assays demonstrated that IiSEP4 interacts with other woad MADS proteins to determine the identity of floral organs. These findings reveal the important roles of IiSEP4 in floral development of I. indigotica. The results of this study can lay a foundation for further study on biological functions of MADS transcriptional factors in I. indigotica.