E. rutaecarpa var. officinalis is a traditional Chinese medicinal plant known for its therapeutic effects, which encompass the promotion of digestion, the dispelling of cold, the alleviation of pain, and the exhibition of anti-inflammatory and antibacterial properties. The principal active component of this plant, limonin, is a potent triterpene compound with notable pharmacological activities. Despite its significance, the complete biosynthesis pathway of limonin in E. rutaecarpa var. officinalis remains incompletely understood, and the underlying molecular mechanisms remain unexplored. The main purpose of this study was to screen the reference genes suitable for expression analysis in E. rutaecarpa var. officinalis, calculate the expression patterns of the genes in the limonin biosynthesis pathway, and identify the relevant enzyme genes related to limonin biosynthesis. The reference genes play a pivotal role in establishing reliable reference standards for normalizing the gene expression data, thereby ensuring precision and credibility in the biological research outcomes. In order to identify the optimal reference genes and gene expression patterns across the diverse tissues (e.g., roots, stems, leaves, and flower buds) and developmental stages (i.e., 17 July, 24 August, 1 September, and 24 October) of E. rutaecarpa var. officinalis, LC-MS was used to analyze the limonin contents in distinct tissue samples and developmental stages, and qRT-PCR technology was employed to investigate the expression patterns of the ten reference genes and eighteen genes involved in limonin biosynthesis. Utilizing a comprehensive analysis that integrated three software tools (GeNorm ver. 3.5, NormFinder ver. 0.953 and BestKeeper ver. 1.0) and Delta Ct method alongside the RefFinder website, the best reference genes were selected. Through the research, we determined that Act1 and UBQ served as the preferred reference genes for normalizing gene expression during various fruit developmental stages, while Act1 and His3 were optimal for different tissues. Using Act1 and UBQ as the reference genes, and based on the different fruit developmental stages, qRT-PCR analysis was performed on the pathway genes selected from the “full-length transcriptome + expression profile + metabolome” data in the limonin biosynthesis pathway of E. rutaecarpa var. officinalis. The findings indicated that there were consistent expression patterns of HMGCR, SQE, and CYP450 with fluctuations in the limonin contents, suggesting their potential involvement in the limonin biosynthesis of E. rutaecarpa var. officinalis. This study lays the foundation for further research on the metabolic pathway of limonin in E. rutaecarpa var. officinalis and provides reliable reference genes for other researchers to use for conducting expression analyses.

Background: Centella asiatica, a medicinally important species that is rich in bioactive compounds, lacks a characterized mitochondrial genome, despite nuclear and chloroplast assemblies. We sequenced and annotated its mitochondrial genome to elucidate its genetic foundations and evolutionary mechanisms. Methods: Assembly using Illumina short-reads and Nanopore long-reads was used to characterize the mitochondrial genome. Analyses included structural characterization, codon usage bias, repetitive sequences, horizontal gene transfer (HGT), collinearity, and phylogeny. The resulting tissue-specific (root, stem, and leaf) long non-coding RNA (lncRNA) profiles identified RNA editing sites. Results: The complete mitochondrial genome (249,777 bp, 45.5% GC) comprises three circular contigs encoding 51 genes (33 protein-coding, 15 tRNA, and 3 rRNA). Comparative genomics revealed synteny with the Apiaceae family of plants and evidence of HGT. Phylogenetic analysis resolved taxonomic relationships within Apiales. We predicted that 547 RNA editing sites would be identified in its protein-coding genes. Tissue profiling identified 725 (root), 711 (stem), and 668 (leaf) editing sites, with >71% concordance to predictions. RNA editing-generated cryptic promoters/terminators occur in mitochondrial core function genes (e.g., ATP synthase, cytochrome c reductase/oxidase, ribosome large subunit, and cytochrome c biogenesis), exhibiting a lower frequency in the leaves compared to the roots and stems. Conclusions: We provide the first complete mitochondrial genome assembly for C. asiatica, delineating its complex structure, tissue-modulated RNA editing, and evolutionary trajectory. This high-quality genomic resource establishes a foundation for molecular evolutionary studies and enhances the genomic toolkit for this pharmacologically significant species.

The size of the mitogenome varies greatly in angiosperms from different species, but the causes of expansion remain unclear. Species from the Zingiberaceae family often carry exceptionally large mitogenomes, where this phenomenon can be readily studied. C. kwangsiensis is a medicinal plant that is native to Guangxi, China, and it is traditionally used to treat blood stasis and gynecological conditions. We assembled its complete mitogenome by using the Illumina and Nanopore reads obtained from its analysis. The genome comprises 12 circular contigs with a multi-branched structure, totaling 7.76 Mb. It contains 39 protein-coding genes, 30 tRNAs (transfer RNAs), three rRNAs (ribosomal RNAs), and 652 C-to-U RNA edited sites. Repeated analysis revealed that the dispersed repeats are the major contributors to genome expansion. Comparisons with two other Zingiberaceae mitogenomes suggested that the large genome arose in a common ancestor rather than from a whole-genome duplication, with little contribution from DNA transfers from the chloroplasts and nuclei. Thus, the exceptionally large mitogenome of Curcuma appears to result largely from repeated accumulation in the non-coding regions, such as nuclear genome expansion in angiosperms. This finding also highlights the conservation of core mitochondrial genes. Our work provides new insights into mitogenomic size variations and gene conservation in plant species, including Zingiberaceae.

Taxillus chinensis (DC.) Danser is an important hemiparasitic medicinal plant whose propagation is severely limited by the desiccation sensitivity of its recalcitrant seeds. Dehydrins (DHNs), which protect plants against dehydration-induced stresses such as salinity, drought, and low temperatures, may play a critical role in protecting recalcitrant seeds. However, the role of DHNs in the seeds of T. chinensis remains unclear. In this study, a differentially expressed gene was identified from the seed transcriptome of T. chinensis and designated TcDHN1. Sequence alignment and phylogenetic analyses revealed that TcDHN1 encodes a dehydrin protein. Heterologous overexpression of TcDHN1 in Arabidopsis did not affect growth under normal conditions. Under salt, drought, and cold stresses, transgenic lines exhibited higher seed germination rates, longer primary roots, and improved seedling growth compared with wild-type (WT) plants. The transgenic lines showed significantly increased activities of antioxidant enzymes, including superoxide dismutase, catalase, and peroxidase. In addition, ectopic overexpression of TcDHN1 in Arabidopsis conferred enhanced tolerance to abiotic stresses compared to WT plants, accompanied by increased expression of the stress-responsive genes Responsive to Desiccation 29A (AtRD29A) and Heat Shock Protein 70-1 (AtHSP70-1). The above results indicate that TcDHN1 confers enhanced tolerance to abiotic stresses. This study provides a functional characterization of an abiotic stress-responsive gene from recalcitrant seeds and identifies a potential genetic resource for molecular breeding. This could potentially improve abiotic stress resistance in T. chinensis and related medicinal plants.