Xiaopu Yin

Curcuma wenyujin (C. wenyujin) is a Dao-di geoherb. It depends on specific ecological conditions. DNA methylation (5mC) mediates environmental stress responses, regulating both growth and bioactive compound synthesis. This implies epigenetic control of secondary metabolism in C. wenyujin. However, its DNA methylation patterns remain uncharacterized. In this study, we identified five CwC5-MTases and three CwdMTases based on the transcriptome of C. wenyujin. They were responsible for DNA methylation and demethylation, respectively. Structural and integrated phylogenetic analysis classified the five CwC5-MTases into four subfamilies: CwMET, CwCMT, CwDRM, CwDNMT. The three CwdMTases were grouped into the ROS subfamily. Both CwC5-MTases and CwdMTases exhibited the closest evolutionary relationship to their homologs in monocots. Treatment of C. wenyujin seedlings with the DNA methyltransferase inhibitor 5-azacytidine (5-Az) enhanced terpenoid biosynthesis. QPCR analysis demonstrated that this treatment significantly upregulated key biosynthetic genes, with the exception of CwDXS. Subsequent GC detection further revealed a concomitant increase in the accumulation of β-elemene. Furthermore, Methylation-Sensitive Amplification Polymorphism (MSAP) analysis revealed that 5-Az altered global DNA methylation patterns. It primarily induced demethylation events. Finally, we explored the nature of these MSAP bands with altered methylation patterns. Gene identification and the effects of 5-Az on terpenoid biosynthesis and methylation not only elucidate the potential role of DNA methylation in secondary metabolism in C. wenyujin but also provide novel insights into the molecular mechanisms underlying its geoherbalism. This research opens a new avenue for breeding high-yield and stress-tolerant cultivars.

Introduction: Curcuma wenyujin (Zingiberaceae), a medicinally significant species within the Curcuma genus, is highly regarded in traditional Chinese medicine for its rich terpenoid constituents. These specialized metabolites serve as the principal bioactive components underpinning the plant's therapeutic effects. As key enzymes in terpenoid biosynthesis, trans -prenyltransferases ( trans -PTs) play crucial roles in regulating metabolic flux. Methods: Trans -PTs in C. wenyujin were identified through a comprehensive transcriptome-wide analysis. The identified CwPTs were classified by constructing a phylogenetic tree. Their transcriptional responses to abscisic acid (ABA), methyl jasmonate (MeJA), and gibberellic acid (GA) were assessed. The catalytic functions of CwFPS1 and CwGGPS1 were characterized through in vitro enzyme assays and heterologous expression in Escherichia coli . Results: Eight trans -CwPTs were identified in C. wenyujin . Among them, five CwPTs function as geranylgeranyl pyrophosphate synthases (GGPS), two as solanesyl pyrophosphate synthases (SPS), and one as farnesyl pyrophosphate synthase (FPS). Expression profile assay showed more significant changes in the transcription levels of Cw PTs in response to ABA and MeJA than to GA. CwFPS1 catalyzed the biosynthesis of farnesyl pyrophosphate (FPP) through the sequential condensation of dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP), as well as geranyl pyrophosphate (GPP) and IPP. CwGGPS1 catalyzed the condensation of GPP or FPP with IPP to generate geranylgeranyl pyrophosphate (GGPP) in vitro . Furthermore, these functions were confirmed in Escherichia coli . Discussion: Our research establishes a molecular foundation for understanding terpenoid biosynthesis in C. wenyujin . Future sequencing of the C. wenyujin genome will facilitate the development of additional CwPTs.

Curcuma wenyujin Y. H. Chen et C. Ling ( C. wenyujin ) is a medicinal plant widely used for clinical treatments. In this study, integrated omics data analyses enabled us to discover the microRNAs (miRNAs) and the phased small interfering RNAs (phasiRNAs) on a transcriptome‐wide scale. A total of 186 mature miRNAs and 23 precursors were reported. Besides, 31 miRNAs of 14 families were organ‐specifically expressed, and 13 of these miRNAs could perform organ‐specific target regulation. More than 80% of the phasiRNA loci were organ‐specifically expressed, especially in tubers. In some cases, phasiRNAs with distinct increments, but with accordant organ‐specific expression patterns, were generated from a highly overlapped region, indicating that different machineries might be synchronously engaged in phasiRNA processing. Based on the transcriptome assembly, 28 and 56 tuber‐specific genes were identified to be involved in alkaloid and terpenoid metabolisms, respectively. Analysis of the enzyme‐coding genes of the β‐elemene biosynthetic pathway showed that the downstream genes were tuber‐specific, while the upstream genes were not. We assumed that the precursor metabolites produced in the other organs might be delivered to the tubers for the final steps of β‐elemene biosynthesis. Summarily, our report provided an organ‐specific transcriptome atlas of C. wenyujin , which could serve as the basis for the molecular studies on organ development and secondary metabolisms in this plant.

β-Elemene (C₁₅H₂₄), a sesquiterpenoid compound isolated from the volatile oil of Curcuma wenyujin, has been proven to be effective for multiple cancers and is widely used in clinical treatment. Unfortunately, the β-elemene content in C. wenyujin is very low, which cannot meet market demands. Our previous research showed that methyl jasmonate (MeJA) induced the accumulation of β-elemene in C. wenyujin. However, the regulatory mechanism is unclear. In this study, 20 jasmonate ZIM-domain (JAZ) proteins in C. wenyujin were identified, which are the core regulatory factors of the JA signaling pathway. Then, the conservative domains, motifs composition, and evolutionary relationships of CwJAZs were analyzed comprehensively and systematically. The interaction analysis indicated that CwJAZs can form homodimers or heterodimers. Fifteen out of twenty CwJAZs were significantly induced via MeJA treatment. As the master switch of the JA signaling pathway, the CwMYC2-like protein has also been identified and demonstrated to interact with CwJAZ2/3/4/5/7/15/17/20. Further research found that the overexpression of the CwMYC2-like gene increased the accumulation of β-elemene in C. wenyujin leaves. Simultaneously, the expressions of HMGR, HMGS, DXS, DXR, MCT, HDS, HDR, and FPPS related to β-elemene biosynthesis were also up-regulated by the CwMYC2-like protein. These results indicate that CwJAZs and the CwMYC2-like protein respond to the JA signal to regulate the biosynthesis of β-elemene in C. wenyujin.

Germacrene A (GA) is a key intermediate for the synthesis of medicinal active compounds, especially for β-elemene, which is a broad-spectrum anticancer drug. The production of sufficient GA in the microbial platform is vital for the precursors supply of active compounds. In this study, Escherichia coli BL21 Star (DE3) was used as the host and cultivated in SBMSN medium, obtaining a highest yield of FPP. The GA synthase from Lactuca sativa (LTC2) exhibited the highest level of GA production. Secondly, two residues involved in product release (T410 and T392) were substituted with Ser and Ala, respectively, responsible for relatively higher activities. Next, substitution of selected residues S243 with Asn caused an increase in activity. Furthermore, I364K-T410S and T392A-T410S were created by combination with the beneficial mutation, and they demonstrated dramatically enhanced titers with 1.90-fold and per-cell productivity with 5.44-fold, respectively. Finally, the production titer of GA reached 126.4 mg/L, and the highest productivity was 7.02 mg/L.h by the I364K-T410S mutant in a shake-flask batch culture after fermentation for 18 h. To our knowledge, the productivity of the I364K-T410S mutant is the highest level ever reported. These results highlight a promising method for the industrial production of GA in E. coli, and lay a foundation for pathway reconstruction and the production of valuable natural sesquiterpenes.

MYB superfamily is one of the most abundant families in plants, and plays critical role in plant growth, development, metabolism regulation, and stress response. Curcuma wenyujin is the main source plant of three traditional Chinese medicines, which are widely used in clinical treatment due to its diverse pharmacological activities. In present study, 88 CwMYBs were identified and analyzed in C. wenyujin, including 43 MYB-related genes, 42 R2R3-MYB genes, two 3R-MYB genes, and one 4R-MYB gene. Forty-three MYB-related proteins were classified into several types based on conserved domains and specific motifs, including CCA1-like type, R-R type, Myb-CC type, GARP-like type, and TBR-like type. The analysis of motifs in MYB DBD and no-MYB regions revealed the relevance of protein structure and function. Comparative phylogeny analysis divided 42 R2R3-MYB proteins into 19 subgroups and provided a reference for understanding the functions of some CwMYBs based on orthologs of previously characterized MYBs. Expression profile analysis of CwMYB genes revealed the differentially expressed genes responding to various abiotic stresses. Four candidate MYB genes were identified by combining the results of phylogeny analysis and expression analysis. CwMYB10, CwMYB18, CwMYB39, and CwMYB41 were significantly induced by cold, NaCl, and MeJA stress treatments. CwMYB18 and CwMYB41 were proved as regulators with activity of transcriptional activation, whereas CwMYB39 and CwMYB10 were not. They may participate in the response to abiotic stresses through different mechanisms in C. wenyujin. This study was the first step toward understanding the CwMYB family and the response to abiotic stresses in C. wenyujin.