Pathway engineering for phenolic acid accumulations in Salvia miltiorrhiza by combinational genetic manipulation.

To produce beneficial phenolic acids for medical and commercial purposes, researchers are interested in improving the normally low levels of salvianolic acid B (Sal B) produced by Salvia miltiorrhiza. Here, we present a strategy of combinational genetic manipulation to enrich the precursors available for Sal B biosynthesis. This approach, involving the lignin pathway, requires simultaneous, ectopic expression of an Arabidopsis Production of Anthocyanin Pigment 1 transcription factor (AtPAP1) plus co-suppression of two endogenous, key enzyme genes: cinnamoyl-CoA reductase (SmCCR) and caffeic acid O-methyltransferase (SmCOMT). Compared with the untransformed control, we achieved a greater accumulation of Sal B (up to 3-fold higher) along with a reduced lignin concentration. This high-Sal B phenotype was stable in roots during vegetative growth and was closely correlated with increased antioxidant capacity for the corresponding plant extracts. Although no outward change in phenotype was apparent, we characterized the molecular phenotype through integrated analysis of transcriptome and metabolome profiling. Our results demonstrated the far-reaching consequences of phenolic pathway perturbations on carbohydrate metabolism, respiration, photo-respiration, and stress responses. This report is the first to describe the production of valuable end products through combinational genetic manipulation in S. miltiorrhiza plants. Our strategy will be effective in efforts to metabolically engineer multi-branch pathway(s), such as the phenylpropanoid pathway, in economically significant medicinal plants.

Overexpression of SmLAC25 promotes lignin accumulation and decreases salvianolic acid content in Salvia miltiorrhiza.

Laccase (LAC) is a blue multicopper oxidase that contains four copper ions, which is involved in lignin polymerization and flavonoid biosynthesis in plants. Although dozens of LAC genes have been identified in Salvia miltiorrhiza Bunge (a model medicinal plant), most have not been functionally characterized. Here, we explored the expression patterns and the functionality of SmLAC25 in S. miltiorrhiza. SmLAC25 has a higher expression level in roots and responds to methyl jasmonate, auxin, abscisic acid, and gibberellin stimuli. The SmLAC25 protein is localized in the cytoplasm and chloroplasts. Recombinant SmLAC25 protein could oxidize coniferyl alcohol and sinapyl alcohol, two monomers of G-lignin and S-lignin. To investigate its function, we generated SmLAC25-overexpressed S. miltiorrhiza plantlets and hairy roots. The lignin content increased significantly in all SmLAC25-overexpressed plantlets and hairy roots, compared with the controls. However, the concentrations of rosmarinic acid and salvianolic acid B decreased significantly in all the SmLAC25-overexpressed lines. Further studies revealed that the transcription levels of some key enzyme genes in the lignin synthesis pathway (e.g., SmCCR and SmCOMT) were significantly improved in the SmLAC25-overexpressed lines, while the expression levels of multiple enzyme genes in the salvianolic acid biosynthesis pathway were inhibited. We speculated that the overexpression of SmLAC25 promoted the metabolic flux of lignin synthesis, which resulted in a decreased metabolic flux to the salvianolic acid biosynthesis pathway.

Genome-wide identification and expression analysis of the superoxide dismutase (SOD) gene family in Salvia miltiorrhiza.

Adverse environmental conditions, such as salinity, cold, drought, heavy metals, and pathogens affect the yield and quality of Salvia miltiorrhiza, a well-known medicinal plant used for the treatment of cardiovascular and cerebrovascular diseases. Superoxide dismutase (SOD), a key enzyme of antioxidant system in plants, plays a vital role in protecting plants against various biotic and abiotic stresses via scavenging the reactive oxygen species produced by organisms. However, little is known about the SOD gene family in S. miltiorrhiza. In this study, eight SOD genes, including three Cu/Zn-SODs, two Fe-SODs and three Mn-SODs, were identified in the S. miltiorrhiza genome. Their gene structures, promoters, protein features, phylogenetic relationships, and expression profiles were comprehensively investigated. Gene structure analysis implied that most SmSODs have different introns/exons distrbution patterns. Many cis-elements related to different stress responses or plant hormones were found in the promoter of each SmSOD. Expression profile analysis indicated that SmSODs exhibited diverse responses to cold, salt, drought, heavy metal, and plant hormones. Additionally, 31 types of TFs regulating SmSODs were predicted and analyzed. These findings provided valuable information for further researches on the functions and applications of SmSODs in S. miltiorrhiza growth and adaptation to stress.

The complete chloroplast genome sequence of Dioscorea zingiberensis (Dioscoreceae).

Dioscorea zingiberensis (Dioscoreceae) is an important medicinal plant endemic to China. Here, its chloroplast genome sequence is reconstructed from the whole-genome Illumina sequencing data. The circular genome is 153,970 bp in length, and comprises a pair of inverted repeat (IR) regions of 25,491 bp each, a large single-copy (LSC) region of 83,950 bp and a small single-copy (SSC) region of 19,038 bp. The chloroplast genome contains 132 genes, including 86 protein-coding genes (79 PCG species), 8 ribosomal RNA genes (four rRNA species) and 38 transfer RNA genes (30 tRNA species). Out of these genes, 10 harbor a single intron, and 7 contain a couple of introns. The overall A + T content of the whole genome is 62.8%, while the corresponding values of the LSC, SSC and IR regions are 64.9%, 68.8% and 57.0%, respectively.

Overexpression of Tomato Prosystemin (LePS) Enhances Pest Resistance and the Production of Tanshinones in Salvia miltiorrhiza Bunge.

Tanshinones are a group of active diterpenes with pharmacological properties that are widely used in the treatment of cardiovascular diseases. Jasmonate (JA) acts as an elicitor to enhance tanshinone biosynthesis in Salvia miltiorrhiza. However, because of high labor costs and undesirable chemical characteristics, the use of JA elicitation is still in the experimental stage. In our experiments, the overexpression of Lycopersicon esculentum (tomato) Prosystemin (LePS) in transgenic plants of S. miltiorrhiza increased their JA concentrations, significantly enhanced the production of tanshinone, and activated the expression of key genes in the tanshinone biosynthesis pathway. Meanwhile, the relative levels of metabolites related to defense such as sterols, terpenes, and phenolic acids were also increased in our OEP lines. In addition, when the larvae of cotton bollworms (Heliothis armigera) were fed with leaves from transgenic lines, their mortality rates rose by nearly 4-fold when compared to that of larvae exposed to leaves from the nontransformed wild type. Our study provides a new strategy for genetic engineering by which tanshinone production and pest resistance can be improved in S. miltiorrhiza. This is accomplished by simulating the wounding signal that increases the endogenous levels of JA.

Effects of total flavonoids from Drynariae Rhizoma prevent bone loss in vivo and in vitro.

Estrogen deficiency is one of the major causes of osteoporosis in postmenopausal women. Drynariae Rhizoma is a widely used traditional Chinese medicine for the treatment of bone diseases. In this study, we investigated the therapeutic effects of the total Drynariae Rhizoma flavonoids (DRTF) on estrogen deficiency-induced bone loss using an ovariectomized rat model and osteoblast-like MC3T3-E1 cells. Our results indicated that DRTF produced osteo-protective effects on the ovariectomized rats in terms of bone loss reduction, including decreased levels of bone turnover markers, enhanced biomechanical femur strength and trabecular bone microarchitecture deterioration prevention. In vitro experiments revealed that the actions of DRTF on regulating osteoblastic activities were mediated by the estrogen receptor (ER) dependent pathway. Our data also demonstrated that DRTF inhibited osteoclastogenesis via up-regulating osteoprotegrin (OPG), as well as down-regulating receptor activator of NF-κB ligand (RANKL) expression. In conclusion, this study indicated that DRTF treatment effectively suppressed bone mass loss in an ovariectomized rat model, and in vitro evidence suggested that the effects were exerted through actions on both osteoblasts and osteoclasts.

Combination of transcriptomic and metabolomic analyses reveals a JAZ repressor in the jasmonate signaling pathway of Salvia miltiorrhiza.

Jasmonates (JAs) are plant-specific key signaling molecules that respond to various stimuli and are involved in the synthesis of secondary metabolites. However, little is known about the JA signal pathway, especially in economically significant medicinal plants. To determine the functions of novel genes that participate in the JA-mediated accumulation of secondary metabolites, we examined the metabolomic and transcriptomic signatures from Salvia miltiorrhiza. For the metabolome, 35 representative metabolites showing significant changes in rates of accumulation were extracted and identified. We also screened out 2131 differentially expressed unigenes, of which 30 were involeved in the phenolic secondary metabolic pathway, while 25 were in the JA biosynthesis and signal pathways. Among several MeJA-induced novel genes, SmJAZ8 was selected for detailed functional analysis. Transgenic plants over-expressing SmJAZ8 exhibited a JA-insensitive phenotype, suggesting that the gene is a transcriptional regulator in the JA signal pathway of S. miltiorrhiza. Furthermore, this transgenic tool revealed that JAZ genes have novel function in the constitutive accumulation of secondary metabolites. Based on these findings, we propose that the combined strategy of transcriptomic and metabolomic analyses is valuable for efficient discovery of novel genes in plants.