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Salvia miltiorrhiza is a model medicinal plant with significant economic and medicinal value. Its roots produce a group of diterpenoid lipophilic bioactive components, termed tanshinones. Biosynthesis and regulation of tanshinones has attracted widespread interest. However, the methylome of S. miltiorrhiza has not been analysed and the regulatory mechanism of DNA methylation in tanshinone production is largely unknown. Here we report single-base resolution DNA methylomes from roots and leaves. Comparative analysis revealed differential methylation patterns for CG, CHG, and CHH contexts and the association between DNA methylation and the expression of genes and small RNAs. Lowly methylated genes always had higher expression levels and 24-nucleotide sRNAs could be key players in the RdDM pathway in S. miltiorrhiza . DNA methylation variation analysis showed that CHH methylation contributed mostly to the difference. Go enrichment analysis showed that diterpenoid biosynthetic process was significantly enriched for genes with downstream overlapping with hypoCHHDMR in July_root when comparing with those in March_root. Tanshinone biosynthesis-related enzyme genes, such as DXS2 , CMK , IDI1 , HMGR2 , DXR , MDS , CYP76AH1, 2OGD25 , and CYP71D373, were less CHH methylated in gene promoters or downstream regions in roots collected in July than those collected in March. Consistently, gene expression was up-regulated in S. miltiorrhiza roots collected in July compared with March and the treatment of DNA methylation inhibitor 5-azacytidine significantly promoted tanshinone production. It suggests that DNA methylation plays a significant regulatory role in tanshinone biosynthesis in S. miltiorrhiza through changing the levels of CHH methylation in promoters or downstreams of key enzyme genes., Competing Interests: The authors declare no conflicts of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nanjing Agricultural University.)

Cytosine DNA methylation is highly conserved epigenetic modification involved in a wide range of biological processes in eukaryotes. It was established and maintained by cytosine-5 DNA methyltransferases (C5-MTases) in plants. Through genome-wide identification, eight putative SmC5-MTase genes were identified from the genome of Salvia miltiorrhiza , a well-known traditional Chinese medicine material and an emerging model medicinal plant. Based on conserved domains and phylogenetic analysis, eight SmC5-MTase genes were divided into four subfamilies, including MET , CMT , DRM and DNMT2 . Genome-wide comparative analysis of the C5-MTase gene family in S. miltiorrhiza and Arabidopsis thaliana , including gene structure, sequence features, sequence alignment and conserved motifs, was carried out. The results showed conservation and divergence of the members of each subfamily in plants. The length of SmC5-MTase open reading frames ranges widely from 1,152 ( SmDNMT2 ) to 5,034 bp ( SmMET1 ). The intron number of SmC5-MTases varies between 7 ( SmDRM1 ) and 20 ( SmCMT1 and SmCMT2b ). These features were similar to their counterparts from Arabidopsis . Sequence alignment and conserved motif analysis showed the existence of highly conserved and subfamily-specific motifs in the C5-MTases analyzed. Differential transcript abundance was detected for SmC5-MTases , implying genome-wide variance of DNA methylation in different organs and tissues. Transcriptome-wide analysis showed that the transcript levels of all SmC5-MTase genes was slightly changed under yeast extract and methyl jasmonate treatments. Six SmC5-MTases , including SmMET1 , SmCMT1 , SmCMT2a , SmCMT2b , SmCMT3 and SmDRM1 , were salicylic acid-responsive, suggesting the involvement of SmC5-MTase s in salicylic acid-dependent immunity. These results provide useful information for demonstrating the role of DNA methylation in bioactive compound biosynthesis and Dao-di herb formation in medicinal plants., Competing Interests: The authors declare there are no competing interests.

Background: FL 32608 Terpenoids are hydrocarbon compounds derived from common fivecarbon isoprene (C5H8) building blocks. They are formed through the condensation and subsequent modification of isoprene units in various ways including - among others - cyclization and/or oxygenation. Their synthesis is localized either to the chloroplast and/or to the cytoplasm/peroxisome/ endoplasmic reticulum and mitochondrion. Terpenoids represent a very large and diverse class of metabolites and play important roles in plant growth and development. In addition, they have been intensively used in human health care, disease treatment and in dietary supplements. Approximately 60% of natural products known so far are terpenoids., Conclusion: This review briefly summarizes the biosynthetic pathways of major plant terpenoids. Then, five well-known and medicinally important diterpenoids, including paclitaxel, tanshinone, ginkgolide, triptolide and oridonin are discussed in detail. Their structures, occurrence, extraction and identification methods, pharmacological properties and clinical uses are also reviewed. Finally, the prospects of using biotechnology to produce medicinally important terpenoids are also briefly discussed., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Small RNA-mediated gene silencing is a vital regulatory mechanism in eukaryotes that requires ARGONAUTE (AGO) proteins. Salvia miltiorrhiza is a well-known traditional Chinese medicinal plant. Therefore, it is important to characterize S. miltiorrhiza AGO family genes as they may be involved in multiple metabolic pathways. This chapter introduces the detailed protocol for SmAGO gene prediction and molecular cloning. In addition, an Agrobacterium-mediated genetic transformation method for S. miltiorrhiza is presented. These methodologies can be used to functionally study SmAGO genes as well as other genes of interest in S. miltiorrhiza.

MicroRNAs (miRNAs) are a specific class of 21-nt small RNAs. They regulate the expression of specific target genes by various types of post-transcriptional regulation mechanisms, such as transcript cleavage and translation suppression. The biological function of an miRNA is therefore intimately associated with the function of their target genes. Target gene identification becomes an essential step towards understanding miRNA functions. In this protocol, we describe a computational procedure for plant miRNA target prediction. It involves two key steps: (1) search of transcript sequence databases for target sequences that have a near-perfect sequence complementarity to the miRNA sequence using the "scan_for_matches" program and (2) evaluation of the miRNA:target sequence pair for pairing complementarity using specific rules, such as positional dependent penalty score and minimum free energy ratio filter, to identify the most likely candidate targets.