The plant Ophiorrhiza pumila produces camptothecin (CPT), a kind of terpene indole alkaloid (TIAs) that has been widely used in treatment of cancer. Tryptophan-arginine-lysine-tyrosine (WRKY) transcription factors have been reported to play important roles in plant metabolism and development. In this study, a novel WRKY transcription factor named OpWRKY3 was isolated from O. pumila, with full-length open reading frame (ORF) of 1128 bp, encoding 375 amino acids. Phylogenetic tree analysis revealed that OpWRKY3 shared the highest homology with VvWRKY30, and it is a significant feature belonging to group III. OpWRKY3 was responsive to various treatments, including gibberellin (GA₃), methyl jasmonate (MJ), acetylsalicylic acid (ASA), salicylic acid (SA), and abscisic acid (ABA). Besides, OpWRKY3 is expressed predominantly in stems. Subcellular localization analysis showed that OpWRKY3 localized in the nucleus. The biomass of OpWRKY3-SRDX transgenic hairy roots (S line) was visibly suppressed, while there were slight changes between overexpression of the OpWRKY3 line (OE line) and the control. In addition, the concentration and total production of camptothecin precursors including loganin and secologanin were significantly changed in both OE and S lines while total production of CPT was significantly changed in most transgenic lines. Thus, the present work revealed that OpWRKY3 may act as a regulator in the growth and development of O. pumila, and in production of camptothecin and its precursors.

Phenolic acids are one of the major secondary metabolites accumulated in Salvia miltiorrhiza with various pharmacological activities. Moderate drought stress can promote the accumulation of phenolic acids in S. miltiorrhiza, while the mechanism remains unclear. Therefore, we performed transcriptome sequencing of S. miltiorrhiza under drought treatment. A total of 47,169 unigenes were successfully annotated in at least one of the six major databases. Key enzyme genes involved in the phenolic acid biosynthetic pathway, including SmPAL, SmC4H, Sm4CL, SmTAT, SmHPPR, SmRAS and SmCYP98A14, were induced. Unigenes annotated as laccase correlated with SmRAS and SmCYP98A14 were analyzed, and seven candidates that may be involved in the key step of SalB biosynthesis by RA were obtained. A total of 15 transcription factors significantly up-regulated at 2 h and 4 h potentially regulating phenolic acid biosynthesis were screened out. TRINITY_DN14213_c0_g1 (AP2/ERF) significantly transactivated the expression of SmC4H and SmRAS, suggesting its role in the regulation of phenolic acid biosynthesis. GO and KEGG enrichment analysis of differential expression genes showed that phenylpropanoid biosynthesis and plant hormone signal transduction were significantly higher. The ABA-dependent pathway is essential for resistance to drought and phenolic acid accumulation. Expression patterns in drought and ABA databases showed that four PYLs respond to both drought and ABA, and three potential SnRK2 family members were annotated and analyzed. The present study presented a comprehensive transcriptome analysis of S. miltiorrhiza affected by drought, which provides a rich source for understanding the molecular mechanism facing abiotic stress in S. miltiorrhiza.

The SnRK gene family is the chief component of plant stress resistance and metabolism through activating the phosphorylation of downstream proteins. S. miltiorrhiza is widely used for the treatment of cardiovascular diseases in Asian countries. However, information about the SnRK gene family of S. miltiorrhiza is not clear. The aim of this study is to comprehensively analyze the SnRK gene family of S. miltiorrhiza and its response to phytohormone. Here, 33 SmSnRK genes were identified and divided into three subfamilies (SmSnRK1, SmSnRK2 and SmSnRK3) according to phylogenetic analysis and domain. SmSnRK genes within same subgroup shared similar protein motif composition and were unevenly distributed on eight chromosomes of S. miltiorrhiza. Cis-acting element analysis showed that the promoter of SmSnRK genes was enriched with ABRE motifs. Expression pattern analysis revealed that SmSnRK genes were preferentially expressed in leaves and roots. Most SmSnRK genes were induced by ABA and MeJA treatment. Correlation analysis showed that SmSnRK3.15 and SmSnRK3.18 might positively regulate tanshinone biosynthesis; SmSnRK3.10 and SmSnRK3.12 might positively regulate salvianolic acid biosynthesis. RNAi-based silencing of SmSnRK2.6 down-regulated the biosynthesis of tanshinones and biosynthetic genes expression. An in vitro phosphorylation assay verified that SmSnRK2.2 interacted with and phosphorylated SmAREB1. These findings will provide a valuable basis for the functional characterization of SmSnRK genes and quality improvement of S. miltiorrhiza.

Introduction:Crocus sativus L. has an important medicinal and economic value in traditional perennial Chinese medicine. However, due to its unique growth characteristics, during cultivation it is highly susceptible to disease. The absence of effective resistance genes restricts us to breed new resistant varieties of C. sativus.Methods: In present study, comprehensive transcriptome sequencing was introduced to explore the disease resistance of the candidate gene in healthy and corm rot-infected C. sativus.Results and discussion: Totally, 43.72 Gb of clean data was obtained from the assembly to generate 65,337 unigenes. By comparing the gene expression levels, 7,575 differentially expressed genes (DEGs) were primarily screened. A majority of the DEGs were completely in charge of defense and metabolism, and 152 of them were annotated as pathogen recognition genes (PRGs) based on the PGRdb dataset. The expression of some transcription factors including NAC, MYB, and WRKY members, changed significantly based on the dataset of transcriptome sequencing. Therefore, this study provides us some valuable information for exploring candidate genes involved in the disease resistance in C. sativus.

Tanshinones, one group of bioactive diterpenes, were widely used in the treatment of cardiovascular diseases. WRKYs play important roles in plant metabolism, but their regulation mechanism in S. miltiorrhiza remains elusive. In this study, one WRKY transcription factor SmWRKY1 was isolated and characterized from S. miltiorrhiza. Multiple sequence alignment and phylogenetic tree analysis showed SmWRKY1 shared high homology with other plant WRKYs such as CrWRKY1. SmWRKY1 were predominantly expressed in leaves and stems, and was responsive to salicylic acid (SA), methyl jasmonate (MeJA) and nitric oxide (NO) treatment. Subcellular localization analysis found that SmWRKY1 was localized in the nucleus. Over-expression of SmWRKY1 significantly elevated the transcripts of genes involved in MEP pathway especially 1-deoxy-D-xylulose 5-phosphate synthase (SmDXS) and 1-deoxy-D-xylulose 5-phosphate reductoisomerase (SmDXR), resulted in over 6 folds increase in tanshinones production in transgenic lines (up to 13.731mg/g dry weight (DW)) compared with the control lines. Dual-luciferase (Dual-LUC) assay showed that SmWRKY1 can positively regulate SmDXR expression by binding to its promoter. Our work revealed that SmWRKY1 participated in the regulation of tanshinones biosynthesis and acted as a positive regulator through activating SmDXR in the MEP pathway, thus discloses a new insight to further excavate the regulation mechanism of tanshinones biosynthesis.