DNA Methylation Influences Chlorogenic Acid Biosynthesis in Lonicera japonica by Mediating LjbZIP8 to Regulate Phenylalanine Ammonia-Lyase 2 Expression

The content of active compounds differ in buds and flowers of Lonicera japonica (FLJ) and L. japonica var. chinensis (rFLJ). Chlorogenic acid (CGAs) were major active compounds of L. japonica and regarded as measurements for quality evaluation. However, little is known concerning the formation of active compounds at the molecular level. We quantified the major CGAs in FLJ and rFLJ, and found the concentrations of CGAs were higher in the buds of rFLJ than those of FLJ. Further analysis of CpG methylation of CGAs biosynthesis genes showed differences between FLJ and rFLJ in the 5′-UTR of phenylalanine ammonia-lyase 2 (PAL2). We identified 11 LjbZIP proteins and 24 rLjbZIP proteins with conserved basic leucine zipper domains, subcellular localization, and electrophoretic mobility shift assay showed that the transcription factor LjbZIP8 is a nuclear-localized protein that specifically binds to the G-box element of the LjPAL2 5′-UTR. Additionally, a transactivation assay and LjbZIP8 overexpression in transgenic tobacco indicated that LjbZIP8 could function as a repressor of transcription. Finally, treatment with 5-azacytidine decreased the transcription level of LjPAL2 and CGAs content in FLJ leaves. These results raise the possibility that DNA methylation might influence the recruitment of LjbZIP8, regulating PAL2 expression level and CGAs content in L. japonica

Full-length transcriptome sequences by a combination of sequencing platforms applied to isoflavonoid and triterpenoid saponin biosynthesis of Astragalus mongholicus Bunge

Abstract Background Astragalus mongholicus Bunge is an important medicinal plant used in traditional Chinese medicine. It is rich in isoflavonoids and triterpenoid saponins. Although these active constituents of A. mongholicus have been discovered for a long time, the genetic basis of isoflavonoid and triterpenoid saponin biosynthesis in this plant is virtually unknown because of the lack of a reference genome. Here, we used a combination of next-generation sequencing (NGS) and single-molecule real-time (SMRT) sequencing to identify genes involved in the biosynthetic pathway of secondary metabolites in A. mongholicus. Results In this study, NGS, SMRT sequencing, and targeted compound analysis were combined to investigate the association between isoflavonoid and triterpenoid saponin content, and specific gene expression in the root, stem, and leaves of A. mongholicus. Overall, 643,812 CCS reads were generated, yielding 121,107 non-redundant transcript isoforms with an N50 value of 2124 bp. Based on these highly accurate transcripts, 104,756 (86.50%) transcripts were successfully annotated by any of the seven databases (NR, NT, Swissprot, KEGG, KOG, Pfam and GO). Levels of four isoflavonoids and four astragalosides (triterpenoid saponins) were determined. Forty-four differentially expressed genes (DEGs) involved in isoflavonoid biosynthesis and 44 DEGs from 16 gene families that encode enzymes involved in triterpenoid saponin biosynthesis were identified. Transcription factors (TFs) associated with isoflavonoid and triterpenoid saponin biosynthesis, including 72 MYBs, 53 bHLHs, 64 AP2-EREBPs, and 11 bZIPs, were also identified. The above transcripts showed different expression trends in different plant organs. Conclusions This study provides important genetic information on the A. mongholicus genes that are essential for isoflavonoid and triterpenoid saponin biosynthesis, and provides a basis for developing the medicinal value of this plant

Molecular Identification and Taxonomic Implication of Herbal Species in Genus Corydalis (Papaveraceae)

Many species of Corydalis (Papaveraceae) have been used as medicinal plants in East Asia, and the most well-known species are Corydalis yanhusuo and C. decumbens in the Pharmacopoeia of China. However, authentication of these species remains problematic because of their high morphological variation. Here, we selected 14 closely related species and five genomic regions (chloroplast: matK, trnG, rbcL, psbA-trnH; nuclear: ITS) to explore the utility of DNA barcoding for authenticating these herbs. In addition, the Poisson tree process (PTP) and automatic barcode gap discovery (ABGD) were also used and compared with DNA barcoding. Our results showed that the ITS region was not suitable for molecular analysis because of its heterogeneous nature in Corydalis. In contrast, matK was an ideal region for species identification because all species could be resolved when matK was used along with the other three chloroplast regions. We found that at least five traditional identified species were lumped into one genetic species by ABGD and PTP methods; thus, highlighting the overestimation of species diversity using the morphological approach. In conclusion, our first attempt of molecular analysis of Corydalis herbs presented here successfully resolved the identification of medicinal species and encouraged their taxonomic re-assessment

Transcriptome-wide identification of WRKY transcription factors and their expression profiles in response to methyl jasmonate in Platycodon grandiflorus

Platycodon grandiflorus, a perennial flowering plant widely distributed in China and South Korea, is an excellent resource for both food and medicine. The main active compounds of P. grandiflorus are triterpenoid saponins. WRKY transcription factors (TFs) are among the largest gene families in plants and play an important role in regulating plant terpenoid accumulation, physiological metabolism, and stress response. Numerous studies have been reported on other medicinal plants; however, little is known about WRKY genes in P. grandiflorus. In this study, 27 PgWRKYs were identified in the P. grandiflorus transcriptome. Phylogenetic analysis showed that PgWRKY genes were clustered into three main groups and five subgroups. Transcriptome analysis showed that the PgWRKY gene expression patterns in different tissues differed between those in Tongcheng City (Southern Anhui) and Taihe County (Northern Anhui). Gene expression analysis based on RNA sequencing and qRT-PCR analysis showed that most PgWRKY genes were expressed after induction with methyl jasmonate (MeJA). Co-expressing PgWRKY genes with triterpenoid biosynthesis pathway genes revealed four PgWRKY genes that may have functions in triterpenoid biosynthesis. Additionally, functional annotation and protein-protein interaction analysis of PgWRKY proteins were performed to predict their roles in potential regulatory networks. Thus, we systematically analyzed the structure, evolution, and expression patterns of PgWRKY genes to provide an important theoretical basis for further exploring the molecular basis and regulatory mechanism of WRKY TFs in triterpenoid biosynthesis

Molecular Cloning and Functional Analysis of Squalene Synthase 2(SQS2) in Salvia miltiorrhiza Bunge

Salvia miltiorrhiza Bunge，which is also known as a traditional Chinese herbal medicine，is widely studied for its ability to accumulate the diterpene quinone Tanshinones. In addition to producing a variety of diterpene quinone, S. miltiorrhiza Bunge also accumulates sterol, brassinosteroid and triterpenoids. During their biosynthesis, squalene synthase (SQS, EC 2.5.1.21) converts two molecules of the hydrophilic substrate farnesyl diphosphate into a hydrophobic product, squalene. In the present study, cloning and characterization of S. miltiorrhiza Bunge squalene synthase 2 (SmSQS2, Genbank Accession Number: KM408605) cDNA was investigated subsequently followed by its recombinant expression and preliminary enzyme activity. The full-length cDNA of SmSQS2 was 1 597 bp in length, with an open reading frame (ORF) of 1 245 bp encoding 414 amino acids. The deduced amino acid sequence of SmSQS2 shared high similarity with those of SQSs from other plants. To obtain soluble recombinant enzymes, the truncated SmSQS2 in which 28 amino acids were deleted from the carboxy terminus was expressed as GST-Tag fusion protein in Escherichia coli BL21 (DE3) and confirmed by SDS-PAGE and Western Blot analysis, and the resultant bacterial crude extract was incubated with farnesyl diphosphate and NADPH. GC-MS analysis showed that squalene was detected in the in vitro reaction mixture. The gene expression level was analyzed through Quantitative real-time PCR, and was found to be higher in roots as compared to the leaves, and was up-regulated upon YE+ Ag+ treatment. These results could serve as an important to understand the function of the SQS family. In addition, the identification of SmSQS2 is important for further studies of terpenoid and sterol biosynthesis in S. miltiorrhiza Bunge

Validation of suitable reference genes for assessing gene expression of MicroRNAs in Lonicera japonica

MicroRNAs (miRNAs), which play crucial regulatory roles in plant secondary metabolism and responses to the environment, could be developed as promising biomarkers for different varieties and production areas of herbal medicines. However, limited information is available for miRNAs from Lonicera japonica, which is widely used in East Asian countries owing to various pharmaceutically active secondary metabolites. Selection of suitable reference genes for quantification of target miRNA expression through quantitative real-time (qRT)-PCR is important for elucidating the molecular mechanisms of secondary metabolic regulation in different tissues and varieties of L. japonica. For precise normalization of gene expression data in L. japonica, 16 candidate miRNAs were examined in three tissues, as well as twenty-one cultivated varieties collected from sixteen production areas, using GeNorm, NormFinder, and RefFinder algorithms. Our results revealed combination of u534122 and u3868172 as the best reference genes across all samples. Their specificity was confirmed by detecting the cycling threshold (Ct) value ranges in different varieties of L. japonica collected from diverse production areas, suggesting the use of these two reference miRNAs is sufficient for accurate transcript normalization with different tissues, varieties, and production areas. To our knowledge, this is the first report on validation of reference miRNAs in honeysuckle (Lonicera spp.). Restuls from this study can further facilitate discovery of functional regulatory miRNAs in different varieties of L. japonica

DataSheet_1_Genome-wide analysis of UDP-glycosyltransferases family and identification of UGT genes involved in drought stress of Platycodon grandiflorus.docx

IntroductionThe uridine diphosphate (UDP)-glycosyltransferase (UGT) family is the largest glycosyltransferase family, which is involved in the biosynthesis of natural plant products and response to abiotic stress. UGT has been studied in many medicinal plants, but there are few reports on Platycodon grandiflorus. This study is devoted to genome-wide analysis of UGT family and identification of UGT genes involved in drought stress of Platycodon grandiflorus (PgUGTs).MethodsThe genome data of Platycodon grandiflorus was used for genome-wide identification of PgUGTs, online website and bioinformatics analysis software was used to conduct bioinformatics analysis of PgUGT genes and the genes highly responsive to drought stress were screened out by qRT-PCR, these genes were cloned and conducted bioinformatics analysis.ResultsA total of 75 PgUGT genes were identified in P.grandiflorus genome and clustered into 14 subgroups. The PgUGTs were distributed on nine chromosomes, containing multiple cis-acting elements and 22 pairs of duplicate genes were identified. Protein-protein interaction analysis was performed to predict the interaction between PgUGT proteins. Additionally, six genes were upregulated after 3d under drought stress and three genes (PGrchr09G0563, PGrchr06G0523, PGrchr06G1266) responded significantly to drought stress, as confirmed by qRT-PCR. This was especially true for PGrchr06G1266, the expression of which increased 16.21-fold after 3d of treatment. We cloned and conducted bioinformatics analysis of three candidate genes, both of which contained conserved motifs and several cis-acting elements related to stress response, PGrchr06G1266 contained the most elements.DiscussionPgGT1 was confirmed to catalyze the C-3 position of platycodin D and only eight amino acids showed differences between gene PGr008G1527 and PgGT1, which means PGr008G1527 may be able to catalyze the C-3 position of platycodin D in the same manner as PgGT1. Seven genes were highly expressed in the roots, stems, and leaves, these genes may play important roles in the development of the roots, stems, and leaves of P. grandiflorus. Three genes were highly responsive to drought stress, among which the expression of PGrchr06G1266 was increased 16.21-fold after 3d of drought stress treatment, indicating that PGrchr06G1266 plays an important role in drought stress tolerance. To summarize, this study laied the foundation to better understand the molecular bases of responses to drought stress and the biosynthesis of platycodin.</p

The Gastrodia elata genome provides insights into plant adaptation to heterotrophy

Gastrodia elata is an obligate mycoheterotrophic plant with highly reduced leaves and bracts in scape. Here, Yuan et al sequence and analyze its 1.06 Gb genome which provides insights in adaptation to a lifestyle of heterotrophy