Medicinal plant microRNAs (miRNAs) are an endogenous class of small RNA central to the posttranscriptional regulation of gene expression. Biosynthetic research has shown that the mature miRNAs in medicinal plants can be produced from either the standard messenger RNA splicing mechanism or the pre-ribosomal RNA splicing process. The medicinal plant miRNA function is separated into two levels: (1) the cross-kingdom level, which is the regulation of disease-related genes in animal cells by oral intake, and (2) the intra-kingdom level, which is the participation of metabolism, development, and stress adaptation in homologous or heterologous plants. Increasing research continues to enrich the biosynthesis and function of medicinal plant miRNAs. In this review, peer-reviewed papers on medicinal plant miRNAs published on the Web of Science were discussed, covering a total of 78 species. The feasibility of the emerging role of medicinal plant miRNAs in regulating animal gene function was critically evaluated. Staged progress in intra-kingdom miRNA research has only been found in a few medicinal plants, which may be mainly inhibited by their long growth cycle, high demand for growth environment, immature genetic transformation, and difficult RNA extraction. The present review clarifies the research significance, opportunities, and challenges of medicinal plant miRNAs in drug development and agricultural production. The discussion of the latest results furthers the understanding of medicinal plant miRNAs and helps the rational design of the corresponding miRNA/target genes functional modules.

Andrographis paniculata is known for its diterpenoid medicinal compounds with antibacterial and anti-inflammatory properties. However, it faces production and cultivation challenges due to low temperatures (LTs). Cytochrome P450 monooxygenases (CYPs) are key enzymes in diterpenoid accumulation. Nevertheless, the functions and LT-related expression patterns of diterpenoid pathway CYPs in Andrographis paniculata remain poorly understood. In this study, 346 CYPs were discovered in Andrographis paniculata. Among them, 328 CYPs belonged to 42 known subfamilies. The remaining 17 CYPs might have represented novel subfamilies unique to this species. A total of 65 candidate CYPs associated with diterpenoid modification were identified. Of these, 50 were transmembrane proteins, and 57 were localized to chloroplasts. The CYP71 subfamily was the most abundant and had the highest motif diversity. Promoters of all candidate CYPs commonly contained elements responsive to gibberellins (GAs), methyl jasmonate (MeJA), and abiotic stresses. Notably, the XP_051152769 protein, corresponding to a CYP gene over 40,000 bp in length, featured an extraordinarily long intron (40,751 nts). Functional elements within this intron were related to LT, GAs, and dehydration pathways. Based on the promoter element arrangement and subfamily classification, 10 representative candidate CYPs were selected. Under LT stress, significant expression changes were observed in three representative CYPs: CYP71D, ent-kaurenoic acid oxidase (KAO), and ent-kaurene oxidase (KO). KAO and KO were significantly upregulated during early LT stress. KAO and KO interacted with each other and jointly interacted with GA20OX2-like. CYP71D acted as a negative response factor to LT stress. Among the 37 proteins interacting with CYP71D, 95% were CYPs. This study provides a critical preliminary foundation for investigating the functions of diterpenoid pathway CYPs in Andrographis paniculata, thereby facilitating the development of LT-tolerant cultivars.