Genome-Wide Identification and Expression Analysis of Sucrose Nonfermenting 1-Related Protein Kinase (SnRK) Genes in Salvia miltiorrhiza in Response to Hormone.

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.

A chromosome-level genome assembly of anesthetic drug-producing Anisodus acutangulus provides insights into its evolution and the biosynthesis of tropane alkaloids.

Tropane alkaloids (TAs), which are anticholinergic agents, are an essential class of natural compounds, and there is a growing demand for TAs with anesthetic, analgesic, and spasmolytic effects. Anisodus acutangulus (Solanaceae) is a TA-producing plant that was used as an anesthetic in ancient China. In this study, we assembled a high-quality, chromosome-scale genome of A. acutangulus with a contig N50 of 7.4 Mb. A recent whole-genome duplication occurred in A. acutangulus after its divergence from other Solanaceae species, which resulted in the duplication of ADC1 and UGT genes involved in TA biosynthesis. The catalytic activities of H6H enzymes were determined for three Solanaceae plants. On the basis of evolution and co-expressed genes, AaWRKY11 was selected for further analyses, which revealed that its encoded transcription factor promotes TA biosynthesis by activating AaH6H1 expression. These findings provide useful insights into genome evolution related to TA biosynthesis and have potential implications for genetic manipulation of TA-producing plants.

Integrated Computing Accelerates Design and Performance Control of New Maraging Steels.

This paper mainly used database technology, machine learning, thermodynamic calculation, experimental verification, etc., on integrated computational materials engineering. The interaction between different alloying elements and the strengthening effect of precipitated phases were investigated mainly for martensitic ageing steels. Modelling and parameter optimization were performed by machine learning, and the highest prediction accuracy was 98.58%. We investigated the influence of composition fluctuation on performance and correlation tests to analyze the influence of elements from multiple perspectives. Furthermore, we screened out the three-component composition process parameters with composition and performance with high contrast. Thermodynamic calculations studied the effect of alloying element content on the nano-precipitation phase, Laves phase, and austenite in the material. The heat treatment process parameters of the new steel grade were also developed based on the phase diagram. A new type of martensitic ageing steel was prepared by selected vacuum arc melting. The sample with the highest overall mechanical properties had a yield strength of 1887 MPa, a tensile strength of 1907 MPa, and a hardness of 58 HRC. The sample with the highest plasticity had an elongation of 7.8%. The machine learning process for the accelerated design of new ultra-high tensile steels was found to be generalizable and reliable.

Heterologous mogrosides biosynthesis in cucumber and tomato by genetic manipulation.

Mogrosides are widely used as high-value natural zero-calorie sweeteners that exhibit an array of biological activities and allow for vegetable flavour breeding by modern molecular biotechnology. In this study, we developed an In-fusion based gene stacking strategy for transgene stacking and a multi-gene vector harbouring 6 mogrosides biosynthesis genes and transformed it into Cucumis sativus and Lycopersicon esculentum. Here we show that transgenic cucumber can produce mogroside V and siamenoside I at 587 ng/g FW and 113 ng/g FW, respectively, and cultivated transgenic tomato with mogroside III. This study provides a strategy for vegetable flavour improvement, paving the way for heterologous biosynthesis of mogrosides.

A jasmonate-responsive bHLH transcription factor TaMYC2 positively regulates triterpenes biosynthesis in Taraxacum antungense Kitag.

Dandelion is a well-known traditional medical herb, also used as functional food. Dandelion possesses many medical properties, such as anti-bacterial and antioxidant activity and contains a variety of triterpenes, such as α-amyrin, ß-amyrin, taraxerol and taraxasterol. In this study, we found that triterpenes biosynthesis was promoted by methyl jasmonate (MeJA), while the transcriptional mechanism underlying triterpenes biosynthesis was rarely investigated. Here, a MeJA-induced bHLH transcription factor TaMYC2 was identified. The content of taraxasterol and taraxerol in dandelion was obviously enhanced in overexpression TaMYC2 transgenic lines and expression level of the squalene synthase gene (TaSS) was elevated to about 3-5 folds compared with the control lines. Dual-LUC, Y1H and EMSA experiments revealed that TaMYC2 bound to the E-box motif in the promoter of TaSS and activated its transcription. Taken together, this study suggested that TaMYC2 acted as a positive regulator for bioengineering approaches to produce high content triterpenes-producing dandelions.

Plant Metabolic Engineering by Multigene Stacking: Synthesis of Diverse Mogrosides.

Mogrosides are a group of health-promoting natural products that extracted from Siraitia grosvenorii fruit (Luo-han-guo or monk fruit), which exhibited a promising practical application in natural sweeteners and pharmaceutical development. However, the production of mogrosides is inadequate to meet the need worldwide, and uneconomical synthetic chemistry methods are not generally recommended for structural complexity. To address this issue, an in-fusion based gene stacking strategy (IGS) for multigene stacking has been developed to assemble 6 mogrosides synthase genes in pCAMBIA1300. Metabolic engineering of Nicotiana benthamiana and Arabidopsis thaliana to produce mogrosides from 2,3-oxidosqualene was carried out. Moreover, a validated HPLC-MS/MS method was used for the quantitative analysis of mogrosides in transgenic plants. Herein, engineered Arabidopsis thaliana produced siamenoside I ranging from 29.65 to 1036.96 ng/g FW, and the content of mogroside III at 202.75 ng/g FW, respectively. The production of mogroside III was from 148.30 to 252.73 ng/g FW, and mogroside II-E with concentration between 339.27 and 5663.55 ng/g FW in the engineered tobacco, respectively. This study provides information potentially applicable to develop a powerful and green toolkit for the production of mogrosides.

A novel WRKY34-bZIP3 module regulates phenolic acid and tanshinone biosynthesis in Salvia miltiorrhiza.

Phenolic acids and tanshinones are main bioactive compounds produced in Salvia miltiorrhiza widely used in treatment of cardiovascular diseases, which could be promoted by abscisic acid elicitation. However, the regulation mechanism remained to be elucidated. An ABA-inducible IIa WRKY transcription factor (TF) named SmWRKY34 exhibiting high homology with AtWRKY40 was isolated. SmWRKY34 exhibited a negative role on phenolic acids and tanshinones by directly regulating SmRAS and SmGGPPS. Moreover, ABA-responsive bZIP TF member named SmbZIP3 expressing significantly in SmWRKY34 transcriptome was screened. SmWRKY34 showed a negative regulatory role on SmbZIP3. SmbZIP3 acted as a positive regulator in the biosynthesis of phenolic acids and tanshinones by targeting SmTAT and two tanshinone-promoting TFs SmERF128 and SmMYB9b. Taken together, we identify a new module WRKY34-bZIP3 involved in ABA signaling that manipulates phenolic acid and tanshinone accumulation, shedding new insights in metabolic engineering application in S. miltiorrhiza.

Heterologous Biosynthesis of Health-Promoting Baicalein in Lycopersicon esculentum.

Baicalein is a valuable flavonoid isolated from the medicinal plant Scutellaria baicalensis Georgi, which exhibits intensive biological activities, such as anticancer and antiviral activities. However, its production is limited in the root with low yield. In this study, In-Fusion and 2A peptide linker were developed to assemble SbCLL-7, SbCHI, SbCHS-2, SbFNSII-2 and SbCYP82D1.1 genes driven by the AtPD7, CaMV 35S and AtUBQ10 promoters with HSP, E9 and NOS terminators, and were used to engineer baicalein biosynthesis in transgenic tomato plants. The genetically modified tomato plants with this construct synthesized baicalein, ranging from 150 ng/g to 558 ng/g FW (fresh weight). Baicalein-fortified tomatoes have the potential to be health-promoting fresh vegetables and provide an alternative source of baicalein production, with great prospects for market application.

Overexpression of SmSCR1 Promotes Tanshinone Accumulation and Hairy Root Growth in Salvia miltiorrhiza.

Lipid-soluble tanshinone is one of the main bioactive substances in the medicinal plant Salvia miltiorrhiza, and its medicinal demand is growing rapidly. Yeast extract (YE) modulates the tanshinone biosynthesis, but the underlying regulatory network remains obscure. In this study, a YE-responsive transcriptional factor Scarecrow1 (SCR1) was identified in S. miltiorrhiza from the YE-induced transcriptome dataset. SmSCR1 is located in the nucleus. Overexpression of SmSCR1 in S. miltiorrhiza roots resulted in a significantly higher accumulation of tanshinone than the control, with the highest 1.49-fold increase. We also detected upregulation of tanshinone biosynthetic genes, SmSCR1 and SmHMGR1, and distinct alteration of growth and development of the hairy roots in the overexpression lines compared to the control. An inverse phenotype was observed in SmSCR1-SRDX suppression expression lines. We found that SmSCR1 can bind to the promoter of SmCPS1 to induce its expression. This study provides new insight into the regulatory mechanism on the growth and development of hairy roots, tanshinone accumulation, and the metabolic engineering of bioactive compounds in S. miltiorrhiza.