Genomic analysis of PIN-FORMED genes reveals the roles of SmPIN3 in root architecture development in Salvia miltiorrhiza.

Salvia miltiorrhiza is a widely utilized medicinal herb in China. Its roots serve as crucial raw materials for multiple drugs. The root morphology is essential for the quality of this herb, but little is known about the molecular mechanism underlying the root development in S. miltiorrhiza. Previous study reveals that the polar auxin transport is critical for lateral root development in S. miltiorrhiza. Whether the auxin efflux carriers PIN-FORMEDs (PINs) are involved in this process is worthy investigation. In this study, we identified nine SmPIN genes in S. miltiorrhiza, and their chromosome localization, physico-chemical properties, and phylogenetic relationship were analyzed. SmPINs were unevenly distributed across four chromosomes, and a variety of hormone responsive elements were detected in their promoter regions. The SmPIN proteins were divided into three branches according to the phylogenetic relationship. SmPINs with close evolutionary distance showed similar conserved motif features. The nine SmPINs showed distinct tissue-specific expression patterns and most of them were auxin-inducible genes. We generated SmPIN3 overexpression S. miltiorrhiza seedlings to investigate the function of SmPIN3 in the root development in this species. The results demonstrated that SmPIN3 regulated the root morphogenesis of S. miltiorrhiza by simultaneously affecting the lateral root development and the root anatomical structure. The root morphology, patterns of root xylem and phloem as well as the expressions of genes in the auxin signaling pathway all altered in the SmPIN3 overexpression lines. Our findings provide new insights for elucidating the regulatory roles of SmPINs in the auxin-mediated root development in S. miltiorrhiza.

Genome-wide identification of the SAUR gene family and screening for SmSAURs involved in root development in Salvia miltiorrhiza.

KEY MESSAGE: SmSAUR4, SmSAUR18, SmSAUR28, SmSAUR37, and SmSAUR38 were probably involved in the auxin-mediated root development in Salvia miltiorrhiza. Salvia miltiorrhiza is a widely utilized medicinal plant in China. Its roots and rhizomes are the main medicinal portions and are closely related to the quality of this herb. Previous studies have revealed that auxin plays pivotal roles in S. miltiorrhiza root development. Whether small auxin-up RNA genes (SAURs), which are crucial early auxin response genes, are involved in auxin-mediated root development in S. miltiorrhiza is worthy of investigation. In this study, 55 SmSAUR genes in S. miltiorrhiza were identified, and their physical and chemical properties, gene structure, cis-acting elements, and evolutionary relationships were analyzed. The expression levels of SmSAUR genes in different organs of S. miltiorrhiza were detected using RNA-seq combined with qRT‒PCR. The root development of S. miltiorrhiza seedlings was altered by the application of indole-3-acetic acid (IAA), and Pearson correlation coefficient analysis was conducted to screen SmSAURs that potentially participate in this physiological process. The diameter of primary lateral roots was positively correlated with SmSAUR4. The secondary lateral root number was positively correlated with SmSAUR18 and negatively correlated with SmSAUR4. The root length showed a positive correlation with SmSAUR28 and SmSAUR37 and a negative correlation with SmSAUR38. The fresh root biomass exhibited a positive correlation with SmSAUR38 and a negative correlation with SmSAUR28. The aforementioned SmSAURs were likely involved in auxin-mediated root development in S. miltiorrhiza. Our study provides a comprehensive overview of SmSAURs and provides the groundwork for elucidating the molecular mechanism underlying root morphogenesis in this species.

DNA methylation regulates biosynthesis of tanshinones and phenolic acids during growth of Salvia miltiorrhiza.

DNA methylation plays a crucial role in the regulation of plant growth and the biosynthesis of secondary metabolites. Danshen (Salvia miltiorrhiza) is a valuable Chinese herbal medicine commonly used to treat cardiovascular diseases; its active ingredients are tanshinones and phenolic acids, which primarily accumulate in roots. Here, we conducted a targeted metabolic analysis of S. miltiorrhiza roots at 3 distinct growth stages: 40 d old (r40), 60 d old (r60), and 90 d old (r90). The contents of tanshinones (cryptotanshinone, tanshinone I, tanshinone IIA, and rosmariquinone) and phenolic acids (rosmarinic acid and salvianolic acid B) gradually increased during plant development. Whole-genome bisulfite sequencing and transcriptome sequencing of roots at the 3 growth stages revealed an increased level of DNA methylation in the CHH context (H represents A, T, or C) context at r90 compared with r40 and r60. Increased DNA methylation levels were associated with elevated expression of various genes linked to epigenetic regulations, including CHROMOMETHYLASE2 (SmCMT2), Decrease in DNA Methylation 1 (SmDDM1), Argonaute 4 (SmAGO4), and DOMAINS REARRANGED METHYLTRANSFERASE 1 (SmDRM1). Moreover, expression levels of many genes involved in tanshinone and salvianolic acid biosynthesis, such as copalyldiphosphate synthase 5 (SmCPS5), cytochrome P450-related enzyme (SmCYP71D464), geranylgeranyl diphosphate synthase (SmGGPPS1), geranyl diphosphate synthase (SmGPPS), hydroxyphenylpyruvate reductase (SmHPPR), and hydroxyphenylpyruvate dioxygenase (SmHPPD), were altered owing to hyper-methylation, indicating that DNA methylation plays an important role in regulating tanshinone and phenolic acid accumulation. Our data shed light on the epigenetic regulation of root growth and the biosynthesis of active ingredients in S. miltiorrhiza, providing crucial clues for further improvement of active compound production via molecular breeding in S. miltiorrhiza.

Progress and prospect: Biosynthesis of plant natural products based on plant chassis.

Plant-derived natural products are a specific class of active substances with numerous applications in the medical, energy, and industrial fields. Many of these substances are in high demand and have become the fundamental materials for various purposes. Recently, the use of synthetic biology to produce plant-derived natural products has become a significant trend. Plant chassis, in particular, offer unique advantages over microbial chassis in terms of cell structure, product affinity, safety, and storage. The development of the plant hairy root tissue culture system has accelerated the commercialization and industrialization of synthetic biology in the production of plant-derived natural products. This paper will present recent progress in the synthesis of various plant natural products using plant chassis, organized by the types of different structures. Additionally, we will summarize the four primary types of plant chassis used for synthesizing natural products from plant sources and review the enabling technologies that have contributed to the development of synthetic biology in recent years. Finally, we will present the role of isolated and combined use of different optimization strategies in breaking the upper limit of natural product production in plant chassis. This review aims to provide practical references for synthetic biologists and highlight the great commercial potential of plant chassis biosynthesis, such as hairy roots.

Comparative transcriptome analysis reveals the regulatory effects of exogenous auxin on lateral root development and tanshinone accumulation in Salvia miltiorrhiza.

MAIN CONCLUSION: The physiological and transcriptome analysis revealed that auxin was a positive regulator of lateral root development and tanshinone accumulation in Salvia miltiorrhiza. Roots of S. miltiorrhiza are widely used as medicinal materials in China, and the root morphology and content of bioactive compounds [such as phenolic acids and diterpenoid quinones (tanshinones)] are the main factors to determine the quality of this herb. Auxin regulates root development and secondary metabolism in many plant species, but little is known about its function in S. miltiorrhiza. In this study, S. miltiorrhiza seedlings were treated (exogenous application) with the auxin indole-3-acetic acid (IAA) and the polar auxin transport inhibitor N-1-naphthylphthalamic acid (NPA) to investigate the regulatory roles of auxin in S. miltiorrhiza. The results indicated that exogenous IAA promoted both lateral root development and tanshinones biosynthesis in S. miltiorrhiza. The NPA application suppressed the lateral root development but showed no obvious effects on tanshinones accumulation. Based on the RNA-seq analysis, expressions of genes related to auxin biosynthesis and signaling transduction were altered in both treated groups. Coincidental with the enhanced content of tanshinones, transcripts of several key enzyme genes in the tanshinones biosynthetic pathway were stimulated after the exogenous IAA application. The expression profiles of seven common transcription factor domain-containing gene families were analyzed, and the results implied that some AP2/ERF genes were probably responsible for the auxin-induced lateral root development in S. miltiorrhiza. These findings shed new light on the regulatory roles of auxin on root development and bioactive compounds biosynthesis in S. miltiorrhiza, and lay the groundwork for future research into the detailed molecular mechanism underlying these biological functions.

Retrospective comparative cohort study of neovagina creation by modified Vecchietti-laparoendoscopic single-site surgery for Mayer-Rokitansky-Küster-Hauser syndrome.

Background: Although traditional multi-incision laparoscopic surgery is minimally invasive, it can still leave surgical scars on the lower abdomen. This study reports a modified Vecchietti-laparoendoscopic single-site (MVLESS) procedure using self-made instruments to create a neovagina for Mayer-Rokitansky-Küster-Hauser syndrome (MRKHS) and to determine the efficacy and safety. Methods: We conducted a retrospective comparative cohort study analysis of the clinical data from MRKHS patients treated in the Department of Difficult Gynecological Disease at Xi'an People's Hospital between January 2010 and December 2020.Women who were diagnosed with MRKHS and underwent a MVLESS procedure using self-made instruments to create a new vagina were selected as the study group (n=14). Patients who underwent a traditional multi-incision Vecchietti (TMV) procedure were selected as the control group (n=22). All participants in both groups were required to complete a Female Sexual Function Index (FSFI) and Female Genital Self-Image Scale (FGSIS) questionnaire. Results: All patients in the study group were diagnosed with type I MRKHS and 3 (21.43%) were sexually active. Twenty-one patients in the control group were diagnosed with type I (95.45%) MRKHS, 1 (4.55%) had type II MRKHS (absent left kidney), and 6 (27.27%) were sexually active. The neovaginas in both groups were wide with good elasticity, softness, smoothness, and lubrication. There were no significant differences in operative time (37±6 vs. 39±4 minutes, P=0.98), intraoperative bleeding (28.32±9.82 vs. 29.45±3.84 mL, P=0.86), postoperative anal exsufflation time (18±4 vs. 20±4 hours, P=0.82), and postoperative hospital stay (7±2 vs. 8±2 days, P=0.84) between the MVLESS and TMV groups. The FSFI and FGSIS scores in both groups showed participants had a good quality of sexual life based on the 12-month follow-up data; however, sexual function was significantly better in the study group than in the control group [lubrication (4.72±0.86 vs. 4.64±1.01, P=0.023), visual analog scale (VAS; 4.26±0.52 vs. 4.45±0.39, P=0.041), total FSFI scores (30.21±4.32 vs. 28.42±2.21, P=0.048), and FGSIS score (23.21±1.98 vs. 22.14±2.04, P=0.012)]. Conclusions: The MVLESS procedure is a simple, safe, and minimally invasive procedure that provides an anatomical and functional neovagina for MRKHS patients. It is an alternative method for creating a neovagina to achieve satisfactory anatomic and sexual function. However, it is still a difficult problem to solve fertility problems for patients with congenital absence of vagina.

Effects of Light on Secondary Metabolite Biosynthesis in Medicinal Plants.

Secondary metabolites (SMs) found in medicinal plants are one of main sources of drugs, cosmetics, and health products. With the increase in demand for these bioactive compounds, improving the content and yield of SMs in medicinal plants has become increasingly important. The content and distribution of SMs in medicinal plants are closely related to environmental factors, especially light. In recent years, artificial light sources have been used in controlled environments for the production and conservation of medicinal germplasm. Therefore, it is essential to elucidate how light affects the accumulation of SMs in different plant species. Here, we systematically summarize recent advances in our understanding of the regulatory roles of light quality, light intensity, and photoperiod in the biosynthesis of three main types of SMs (polyphenols, alkaloids, and terpenoids), and the underlying mechanisms. This article provides a detailed overview of the role of light signaling pathways in SM biosynthesis, which will further promote the application of artificial light sources in medicinal plant production.

[Roles of reactive oxygen species in Streptomyces pactum Act12-induced tanshinone production in Salvia miltiorrhiza hairy roots].

Our previous research indicated that the Streptomyces pactum Act12 (Act12) had a certain promotional effect on tanshinone accumulation and up-regulated the expression of genes 3-hydroxy-3-methyglutaryl-CoA reductase (HMGR) and 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR) in Salvia miltiorrhiza hairy roots. This study focuses on the roles of reactive oxygen species in S. pactum Act12-induced tanshinone production in S. miltiorrhiza hairy roots. The 4% Act12, 4% Act12 + CAT and 4% Act12 + SOD were added to S. miltiorrhiza hairy root and subcultured for 21 days, the dry weight, contents of reactive oxygen species, contents of tanshinones and expression of HMGR and DXR were determined at different harvest-time. The generation of reactive oxygen species (ROS) in S. miltiorrhiza hairy roots was triggered by 4% Act12 treatment. The relative expressions of genes HMGR and DXR in 4% Act12 treatment were 32.4 and 4.8-fold higher than those in the control. And the total tanshinone in the hairy roots was 10.2 times higher than that of the control. The CAT and SOD could significantly inhibit the ROS accumulation and relative expressions of genes HMGR and DXR in 4% Act12 treatment, which induced the total tanshinone content was decreased by 74.6% comparing with the 4% Act12 treatment. ROS mediated Act12-induced tanshinone production. The Act12 may be via the ROS signal channel to activate the tanshinone biosynthesis pathways. Thereby the tanshinon content in hairy roots was increased.

Effects of Streptomyces pactum Act12 on Salvia miltiorrhiza hairy root growth and tanshinone synthesis and its mechanisms.

The fermentation broth and mycelium pellet of Streptomyces pactum Act12 (Act12) may promote the accumulation of soluble sugar when added to Salvia miltiorrhiza hairy roots, increasing the accumulation level by as much as 23.20 % compared with the control; it may also inhibit the accumulation of soluble protein in the hairy roots, decreasing it by as much as 17.96 % compared with the control. The ACT12 also has a certain promotional effect on the growth of hairy root at an appropriate concentration of elicitors and upregulates the expression of genes 3-hydroxy-3-methyglutary1-CoA reductase (HMGR), 1-deoxy-D-xylulose 5-phosphate synthase (DXS), 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR), and geranylgeranyl diphosphate synthase (GGPPS). Among these effects, that of the HMGR gene expression is as high as 33.66 times that of the control, indicating that the test Streptomyces pactum may efficiently adjust the secondary metabolism of S. miltiorrhiza at the level of gene transcription, thereby greatly increasing the accumulation level of tanshinone in the hairy roots; among which, the cryptotanshinone levels increased most significantly, as much as 33.63 times that of the control, and the total tanshinone levels were 12.61 times that of the control.

Selective responses of enzymes in the two parallel pathways of rosmarinic acid biosynthetic pathway to elicitors in Salvia miltiorrhiza hairy root cultures.

Rosmarinic acid and salvianolic acid B are two important phenolic compounds with therapeutic properties in Salvia miltiorrhiza Bunge. The biosynthesis of rosmarinic acid is initiated by two parallel pathways, namely the phenylpropanoid pathway and the tyrosine-derived pathway. Salvianolic acid B is a structural dimer of rosmarinic acid and is believed to be derived from rosmarinic acid. In the current study, methyl jasmonate (MeJA) and hyphal extracts from fungi were used as elicitors to examine the relationship between enzymes in the two parallel pathways and accumulation of phenolic compounds in S. miltiorrhiza hairy root cultures. The results showed that accumulations of rosmarinic acid, salvianolic acid B and total phenolics were enhanced by MeJA while suppressed by fugal extracts. Responses of enzymes in the tyrosine-derived pathway, at both the gene transcript and enzyme activity levels, showed a better consistency with alterations of phenolic compounds content after the two elicitors treated. Our study implied that compared with enzymes in the phenylpropanoid pathway, enzymes in the tyrosine-derived pathway are more correlated to rosmarinic acid and salvianolic acid B biosynthesis in S. miltiorrhiza hairy roots.

Cloning and characterization of a putative R2R3 MYB transcriptional repressor of the rosmarinic acid biosynthetic pathway from Salvia miltiorrhiza.

Salvia miltiorrhiza Bunge is one of the most renowned traditional medicinal plants in China. Phenolic acids that are derived from the rosmarinic acid pathway, such as rosmarinic acid and salvianolic acid B, are important bioactive components in S. miltiorrhiza. Accumulations of these compounds have been reported to be induced by various elicitors, while little is known about transcription factors that function in their biosynthetic pathways. We cloned a subgroup 4 R2R3 MYB transcription factor gene (SmMYB39) from S. miltiorrhiza and characterized its roles through overexpression and RNAi-mediated silencing. As the results showed, the content of 4-coumaric acid, rosmarinic acid, salvianolic acid B, salvianolic acid A and total phenolics was dramatically decreased in SmMYB39-overexpressing S. miltiorrhiza lines while being enhanced by folds in SmMYB39-RNAi lines. Quantitative real-time PCR and enzyme activities analyses showed that SmMYB39 negatively regulated transcripts and enzyme activities of 4-hydroxylase (C4H) and tyrosine aminotransferase (TAT). These data suggest that SmMYB39 is involved in regulation of rosmarinic acid pathway and acts as a repressor through suppressing transcripts of key enzyme genes.

[Effects of elicitors on accumulation of phenolic acids and tanshinones in Salvia miltiorrhiza hairy root].

OBJECTIVE: To observe the effects of a biotic elicitor fungal hyphae extract, an abiotic elicitor methyl jasmonate and their synergistic action on the accumulation of phenolic acids and tanshinones in Salvia miltiorrhiza hairy root. METHOD: Different elicitors were added to S. miltiorrhiza hairy root, which was subcultured for 21 days, the dry weight and contents of phenolic acids and tanshinones were determined at different harvest-time. RESULT: S. miltiorrhiza hairy root growth was significantly inhibited by all three treatments and the accumulation of cryptotanshinone and dihydrotanshinone were promoted by each elicition. As for the accumulation of phenolic acids, there were differences between fungal elicitor and methyl jasmonate treatments, they were promoted by methyl jasmonate while inhibited in a certain extent by fungal hyphae extract. CONCLUSION: Fungal elicitor, methyl jasmonate and their synergistic action have significant influence on accumulation of components in S. miltiorrhiza hairy root, and the effect varies between phenolic acids and tanshinones. There is no correlation between production of water-soluble ingredients and fat-soluble components on the whole under three different treatments.