Impact of Methyl Jasmonate on Terpenoid Biosynthesis and Functional Analysis of Sesquiterpene Synthesis Genes in Schizonepeta tenuifolia.

This study investigates the impact of methyl jasmonate (MeJA) on the volatile oil composition of Schizonepeta tenuifolia and elucidates the function of the StTPS45 gene, a key player in terpenoid biosynthesis. The effect of different concentrations of MeJA (0, 50, 100, 200, and 300 µmol/L) on the growth of S. tenuifolia adventitious bud clusters was analyzed over a 20 d period. Using gas chromatography-mass spectrometry (GC-MS), 17 compounds were identified from the adventitious bud clusters of S. tenuifolia. Significant changes in the levels of major monoterpenes, including increased contents of (+)-limonene and (+)-menthone, were observed, particularly at higher concentrations of MeJA. Analysis of transcriptome data from three groups treated with 0, 100, and 300 µmol/L MeJA revealed significant changes in the gene expression profiles following MeJA treatment. At 100 µmol/L MeJA, most terpene synthase (TPS) genes were overexpressed. Additionally, gene expression and functional predictions suggested that StTPS45 acts as germacrene D synthase. Therefore, StTPS45 was cloned and expressed in Escherichia coli, and enzyme activity assays confirmed its function as a germacrene D synthase. Molecular docking and structural prediction of StTPS45 further suggested specific interactions with farnesyl diphosphate (FPP), aligning with its role in the terpenoid synthesis pathway. These findings provide valuable insights into the modulation of secondary metabolite pathways by jasmonate signaling and underscore the potential of genetic engineering approaches to enhance the production of specific terpenoids in medicinal plants.

Transcription factor StWRKY1 is involved in monoterpene biosynthesis induced by light intensity in Schizonepeta tenuifolia Briq.

Menthone-type monoterpenes are the main active ingredients of Schizonepeta tenuifolia Briq. Previous studies have indicated that light intensity influences the synthesis of menthone-type monoterpenes in S. tenuifolia, but the mechanism remains unclear. WRKY transcription factors play a crucial role in plant metabolism, yet their regulatory mechanisms in S. tenuifolia are not well understood. In this study, transcriptome data of S. tenuifolia leaves under different light intensities were analyzed, identifying 57 candidate transcription factors that influence monoterpene synthesis. Among these, 7 members of the StWRKY gene family were identified and mapped onto chromosomes using bioinformatics methods. The physicochemical properties of the proteins encoded by these StWRKY genes, their gene structures, and cis-acting elements were also studied. Comparative genomics and phylogenetic analyses revealed that Sch000013479 is closely related to AaWRKY1, AtWRKY41, and AtWRKY53, and it was designated as StWRKY1. Upon silencing and overexpressing the StWRKY1 transcription factor in S. tenuifolia leaves, changes in the expression of key genes in the menthone-type monoterpene synthesis pathway were observed. Specifically, when StWRKY1 was effectively silenced, the content of (-)-pulegone significantly decreased. These results enhance our understanding of the impact of StWRKYs on monoterpene synthesis in S. tenuifolia and lay the groundwork for further exploration of the regulatory mechanisms involved in the biosynthesis of menthone-type monoterpenes.

Heat Stress and Microbial Stress Induced Defensive Phenol Accumulation in Medicinal Plant Sparganium stoloniferum.

An approach based on the heat stress and microbial stress model of the medicinal plant Sparganium stoloniferum was proposed to elucidate the regulation and mechanism of bioactive phenol accumulation. This method integrates LC-MS/MS analysis, 16S rRNA sequencing, RT-qPCR, and molecular assays to investigate the regulation of phenolic metabolite biosynthesis in S. stoloniferum rhizome (SL) under stress. Previous research has shown that the metabolites and genes involved in phenol biosynthesis correlate to the upregulation of genes involved in plant-pathogen interactions. High-temperature and the presence of Pseudomonas bacteria were observed alongside SL growth. Under conditions of heat stress or Pseudomonas bacteria stress, both the metabolites and genes involved in phenol biosynthesis were upregulated. The regulation of phenol content and phenol biosynthesis gene expression suggests that phenol-based chemical defense of SL is stimulated under stress. Furthermore, the rapid accumulation of phenolic substances relied on the consumption of amino acids. Three defensive proteins, namely Ss4CL, SsC4H, and SsF3'5'H, were identified and verified to elucidate phenol biosynthesis in SL. Overall, this study enhances our understanding of the phenol-based chemical defense of SL, indicating that bioactive phenol substances result from SL's responses to the environment and providing new insights for growing the high-phenol-content medicinal herb SL.

[Accumulation and biosynthesis mechanism of volatile oils in glandular scale of Agastache rugosa].

The volatile oils are the effective components of Agastache rugosa, which are stored in the glandular scale. The leaves of pulegone-type A. rugosa were used as materials to observe the leaf morphology of A. rugosa at different growth stages, and the components of volatile oils in gland scales were detected by GC-MS. At the same time, qRT-PCR was used to determine the relative expression of key enzyme genes in the biosynthesis pathway of monoterpenes in volatile oils. The results showed that the density of A. rugosa glandular scale decreased first and then tended to be stable. With the growth of leaves, the relative content of pulegone decreased from 79.26% to 3.94%(89.97%-41.44%), while that of isomenthone increased from 2.43% to 77.87%(0.74%-51.01%), and the changes of other components were relatively insignificant. The correlation analysis between the relative content of monoterpenes and the relative expression levels of their key enzyme genes showed that there was a significant correlation between the relative content of menthone and isomenthone and the relative expression levels of pulegone reductase(PR)(r>0.6, P<0.01). To sum up, this study revealed the accumulation rules of the main components of the contents of the glandular scale of A. rugosa and the expression rules of the key enzyme genes for biosynthesis, which provided a scientific basis and data support for determining the appropriate harvesting period and quality control of the medicinal herbs. This study also initially revealed the biosynthesis mechanism of the monoterpenes mainly composed of pulegone and isomenthone in A. rugosa, laying a foundation for further research on the molecular mechanism of synthesis and accumulation of monoterpenes in A. rugosa.

[Identification and analysis of terpene synthase (TPS) gene family in Schizonepeta tenuifolia].

Terpenoids are important secondary metabolites of plants that possess both pharmacological activity and economic value. Terpene synthases(TPSs) are key enzymes in the synthesis process of terpenoids. In order to investigate the TPS gene family members and their potential functions in Schizonepeta tenuifolia, this study conducted a systematic analysis of the TPS gene family of S. tenuifolia based on the whole genome data of S. tenuifolia using bioinformatics methods. The results revealed 57 StTPS members identified from the genome database of S. tenuifolia. The StTPS family members encoded 285-819 amino acids, with protein molecular weights ranging from 32.75 to 94.11 kDa, all of which were hydrophilic proteins. The StTPS family members were mainly distributed in the cytoplasm and chloroplasts, exhibiting a random and uneven physical localization pattern. Phylogenetic analysis showed that the StTPS genes family were divided into six subgroups, mainly belonging to the TPS-a and TPS-b subfamilies. Promoter analysis predicted that the TPS gene family members could respond to various stressors such as light, abscisic acid, and methyl jasmonate(MeJA). Transcriptome data analysis revealed that most of the TPS genes were expressed in the roots of S. tenuifolia, and qRT-PCR analysis was conducted on genes with high expression in leaves and low expression in roots. Through the analysis of the TPS gene family of S. tenuifolia, this study identified StTPS5, StTPS18, StTPS32, and StTPS45 as potential genes involved in sesquiterpene synthesis of S. tenuifolia. StTPS45 was cloned for the construction of an prokaryotic expression vector, providing a reference for further investigation of the function and role of the TPS gene family in sesquiterpene synthesis.

Addition of venetoclax to myeloablative conditioning regimens for allogeneic hematopoietic stem cell transplantation in high-risk AML.

BACKGROUND: Venetoclax monotherapy is an effective option for patients with acute myeloid leukemia (AML). Venetoclax has also been used in non-myeloablative conditioning allogeneic hematopoietic stem cell transplantation (allo-HSCT) for high-risk AML with a tolerable toxicity profile. However, the efficacy and safety of a venetoclax-containing myeloablative conditioning (MAC) allo-HSCT regimen for high-risk AML have not been evaluated. OBJECTIVE: To evaluate the safety and efficacy of a MAC regimen containing venetoclax for high-risk AML. STUDY DESIGN: From 25 February 2021 to 4 September 2022, a total of 31 patients with high-risk AML who underwent allo-HSCT and a MAC regimen with venetoclax were analyzed. RESULTS: At the time of transplantation, 21 patients were in first complete remission (CR1), 4 were in a second complete remission (CR2), and 6 in non-remission (NR). Twenty-four patients (77.4%) were minimal residual disease (MRD)-positive before transplant. The FLT3-ITD gene mutation was present in 51.6% of patients. NUP98 rearrangement, MLL rearrangement or MLL-PTD and DEK::CAN fusion genes were found in 5 (16.1%), 7(22.6%) and 2 (6.5%) patients, respectively. Twenty-nine (93.6%) patients underwent haploidentical allo-HSCT. The median follow-up time was 278 days (range: 52-632 days). The 100-day cumulative incidence of grade 3 to 4 acute graft-versus-host disease (aGVHD) was 16.1% (95%CI, 7.2-36.0%). The 180-day cumulative incidence of moderate to severe chronic graft-versus-host disease (cGVHD) was 7.1% (95%CI, 1.9-26.9%). Cumulative incidence of 100-day cytomegalovirus (CMV) viraemia and 100-day Epstein-Barr virus (EBV) viraemia was 61.6% (95%CI, 46.5-81.4%) and 3.2% (95%CI, 0.4-22.2%), respectively. The 600-day overall survival (OS) and leukemia-free survival (LFS) were 80.9% (95%CI, 63.5-93.6%) and 81.3% (95%CI, 64.2-93.7%), respectively. The 600-day relapse incidence (RI) and non-relapse mortality (NRM) was 6.9% (95%CI, 1.8-26.3%) and 11.7% (95%CI, 3.9-35.0%). CONCLUSION: Our study shows that the addition of venetoclax to a MAC allo-HSCT was feasible, safe and effective for high-risk AML patients.

A chromosome-level genome assembly reveals that a bipartite gene cluster formed via an inverted duplication controls monoterpenoid biosynthesis in Schizonepeta tenuifolia.

Biosynthetic gene clusters (BGCs) are regions of a genome where genes involved in a biosynthetic pathway are in proximity. The origin and evolution of plant BGCs as well as their role in specialized metabolism remain largely unclear. In this study, we have assembled a chromosome-scale genome of Japanese catnip (Schizonepeta tenuifolia) and discovered a BGC that contains multiple copies of genes involved in four adjacent steps in the biosynthesis of p-menthane monoterpenoids. This BGC has an unprecedented bipartite structure, with mirrored biosynthetic regions separated by 260 kilobases. This bipartite BGC includes identical copies of a gene encoding an old yellow enzyme, a type of flavin-dependent reductase. In vitro assays and virus-induced gene silencing revealed that this gene encodes the missing isopiperitenone reductase. This enzyme evolved from a completely different enzyme family to isopiperitenone reductase from closely related Mentha spp., indicating convergent evolution of this pathway step. Phylogenomic analysis revealed that this bipartite BGC has emerged uniquely in the S. tenuifolia lineage and through insertion of pathway genes into a region rich in monoterpene synthases. The cluster gained its bipartite structure via an inverted duplication. The discovered bipartite BGC for p-menthane biosynthesis in S. tenuifolia has similarities to the recently described duplicated p-menthane biosynthesis gene pairs in the Mentha longifolia genome, providing an example of the convergent evolution of gene order. This work expands our understanding of plant BGCs with respect to both form and evolution, and highlights the power of BGCs for gene discovery in plant biosynthetic pathways.

[Cloning of StHD1 and StHD8 from Schizonepeta tenuifolia and function of regulating glandular trichome development].

Hd-Zip, a unique transcription factor in plant kingdom, influences the growth, development, and secondary metabolism of plants. Hd-zip Ⅳ is thought to play an important role in trichome development of Schizonepeta tenuifolia. This study aims to explore the functions of StHD1 and StHD8 in Hd-zip Ⅳ subfamily in peltate glandular trichome development. To be specific, the expression patterns of the two genes and interaction between the proteins encoded by them were analyzed based on transcriptome sequencing and two-hybrid screening. The subcellular localization was performed and functions of the genes were verified in tobacco and S. tenuifolia. The results showed that StHD1 and StHD8 had high similarity to HD-Zip Ⅳ proteins of other plants and they all had the characteristic conserved domains of HD-Zip Ⅳ subfamily. They were located in the nucleus. The two genes mainly expressed in young tissues and spikes, and StHD1 and StHD8 proteins interacted with each other. The density and length of glandular trichomes increased significantly in tobacco plants with the overexpression of StHD1 and StHD8. Inhibiting the expression of StHD1 and StHD8 by VIGS(virus-induced gene silencing) in S. tenuifolia resulted in the reduction in the density of peltate glandular trichomes, the expression of key genes related to mono-terpene synthesis, and the relative content of limonene and pulegone, the main components of monoterpene. These results suggested that StHD1 and StHD8 of S. tenuifolia formed a complex to regulate glandular trichomes and affect the biosynthesis of monoterpenes.

Single-cell transcriptome of Nepeta tenuifolia leaves reveal differentiation trajectories in glandular trichomes.

The peltate glandular trichomes (PGTs) on Nepeta tenuifolia leaves can secrete and store bioactive essential oils. ScRNA-seq is a powerful tool for uncovering heterogeneous cells and exploring the development and differentiation of specific cells. Due to leaves rich in PGTs, the young leaves were used to isolated protoplasts and successfully captured 33,254 protoplasts for sequencing purposes. After cell type annotation, all the cells were partitioned into six broad populations with 19 clusters. Cells from PGTs were identified based on the expression patterns of trichome-specific genes, monoterpene biosynthetic genes, and metabolic analysis of PGT secretions. The developmental trajectories of PGTs were delineated by pseudotime analysis. Integrative analysis of scRNA-seq data from N. tenuifolia leaves and Arabidopsis thaliana shoot revealed that PGTs were specific to N. tenuifolia. Thus, our results provide a promising basis for exploring cell development and differentiation in plants, especially glandular trichome initiation and development.

Identification and characterization of a novel gene involved in glandular trichome development in Nepeta tenuifolia.

Nepeta tenuifolia is a medicinal plant rich in terpenoids and flavonoids with antiviral, immunoregulatory, and anti-inflammatory activities. The peltate glandular trichome (PGT) is a multicellular structure considered to be the primary storage organ for monoterpenes; it may serve as an ideal model for studying cell differentiation and the development of glandular trichomes (GTs). The genes that regulate the development of GTs have not yet been well studied. In this study, we identified NtMIXTA1, a GT development-associated gene from the R2R3 MYB SBG9 family. NtMIXTA1 overexpression in tobacco resulted in the production of longer and denser GTs. Virus-induced gene silencing of NtMIXTA1 resulted in lower PGT density, a significant reduction in monoterpene concentration, and the decreased expression of genes related to monoterpene biosynthesis. Comparative transcriptome and widely targeted metabolic analyses revealed that silencing NtMIXTA1 significantly influenced the expression of genes, and the production of metabolites involved in the biosynthesis of terpenoids, flavonoids, and lipids. This study provides a solid foundation describing a mechanism underlying the regulation of GT development. In addition, this study further deepens our understanding of the regulatory networks involved in GT development and GT development-associated metabolite flux, as well as provides valuable reference data for studying plants with a high medicinal value without genetic transformation.